1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, 그리고 세포 내 활성 산소종에 의한 라디칼 소거율을 측정하였더니 esculetin 에 의해 DPPH radical 제거, hydroxyl radical 제거와 세포 내 활성 산소종 제거 능력을 보였다. 지방 과산화의 척도인 thiobarbituric acid 와 반응하는 물질을 측정한 결과 esculetin 이 지방 과산화를 방지함을 알 수 있었다. Protein carbonyl ELISA kit 를 이용하여 단백질 과산화의 척도인 carbonyl formation 을 측정한 결과 esculetin 에 의해 단백질에서 carbonyl formation 도 방지됨을 확인하였다. 또한, western blot 과 면역형광상을 통해 세포 DNA 손상을 측정한 결과 esculetin 을
Although many studies have reported the protectiveeffectof flavonoids including baicalein againstoxidativestress-inducedcelldamage, few have examined the effects on cellular repair mechanisms mediated by flavonoids. Therefore, in this study, we evaluated the effects of baicalein on cellular DNA repair responses to oxidativestress. Baicalein has free radical scavenging and antioxidant effects due to the trihydroxy structure of its A ring  and its lipophilic characteristics . In our study, baicalein effectively reduced ROS and suppressed DNA breakage in response to oxidativestressvia the induction of Ku70 and DNA-PKcs, demonstrating the ability of baicalein to promote the repair of DSBs.
3. Effect of hyperoside on apoptosis induced by H 2 O 2
The protectiveeffectof hyperoside on cell survival in H 2 O 2 treated cells was measured.
Cells were treated with hyperoside at 5 mM for 1 h, prior to the addition of H 2 O 2 . Cell viability was determined 24 h later by the MTT assay. As shown in Fig. 6, treatment with hyperoside increased the cell survival by 56% as compared to 42% of H 2 O 2 treatment. To evaluate a cytoprotective effectof hyperoside on apoptosis induced by H 2 O 2 , the nuclei of V79-4 cells were stained with Hoechst 33342 and assessed by microscopy. The microscopic pictures in Fig. 7A showed that the control cells had intact nuclei, while H 2 O 2 treated cells showed significant nuclear fragmentation, which is characteristic of apoptosis. When the cells were treated with hyperoside for 1 h prior to H 2 O 2 treatment, however, a decrease in nuclear fragmentation was observed. In addition to the morphological evaluation, the protectiveeffectof hyperoside against apoptosis was also confirmed by apoptotic sub-G 1
Protective effectof methanol extract on H 2 O 2 -inducedoxidativedamage
In order to study celldamageprotectiveeffectof a medicinal plant, B. platyphylla var. japonica total methanol extract, we treated cells with chemical and physical stress which cause cellular damage. First of all, in order to determine appropriate H 2 O 2 concentration, H 2 O 2 dose- dependent cell proliferation was measured. Cell proliferation was gradually decreased as the concentration of H 2 O 2 increases (Fig. 1A). Approximately 50% ofcell proliferation was observed at 70 µM of H 2 O 2 . When cells were pre-treated with total extract before treatment of 100 µM of H 2 O 2 , the cell proliferation of H 2 O 2 treated cells was increased as dose-dependent manner of total extract treated (Fig. 1B); 106.9 %, 122.1% and 173.7% ofcell proliferation at 4, 20 and 100 µg/ml of total extract, respectively.
。 xygen species generated by irradiation. Our data demonstrate that dieckol produces a radioprotective effect
。 n V79-4 lung fibrob1ast cells. which are known ω be sensitive ω irradiation π113protective mechanism of dieckol invo1ves its ability to scavenge ROS. Jn màl1Ycases ‘ the y- ray radiation-inducedcell death has resulted in apopωsis6 24). Diecko1 increased cell survival vîa inhibition of )'-ray radiation-induced apoptosis. This cytoprotective effectinduced by dieckol was associated with the inhibited expression of phospho-H2A.X and caspase 9 activity. Taken together. the radioprotective efTect of diecko1 against y-ray radiation-inducedcelldamage was exerted via ROS scavenging acti 찌 ty
한 C6 glial cell 보호 효능을 확인하고자 하였다. In vitro assay에서 G. uralensis, G. glabra, SW 추출물은 농도유의적으 로 2,2-diphenyl-1-picrylhydrazyl, ·OH, O 2 − radical 소거능이 증 가하여 in vitro 항산화 활성을 확인하였다. 또한, SW 추출물은 G. uralensis, G. glabra 추출물에 비해 총 페놀 및 플라보노이 드 함량이 가장 우수하였다. H 2 O 2 로 산화적 스트레스를 유도한 C6 glial cell에 3가지 감초 추출물을 각각 처리 시, 농도의존적 으로 세포 생존율이 증가와 reactiveoxygenspecies 소거능이 증가하여 3가지 감초 추출물의 산화적 손상에 대한 신경교세포 보호 효과를 확인하였다. 특히, SW 추출물은 G. uralensis, G.
Ultraviolet B (UVB) radiation causes skin diseases by oxidativestress. This study investigated the protectiveeffectof mackerel-derived fermented fish oil (FFO) against UVB radiation-inducedoxidativestress in human HaCaT keratinocytes and mouse skin tissue. HaCaT cells were exposed to 30 mJ/cm 2 of UVB radiation for 24h with or without FFO treatment, which showed cytoprotective effects by scavenging UVB-induced intracellular reactiveoxygenspecies. FFO treatment attenuated UVB-inducedoxidative modifications including lipid peroxidation, protein carbonylation, and DNA damage. FFO treatment also reduced UVB-induced apoptosis by reducing apoptotic bodies, DNA fragmentation, caspase activation ,and proapoptotic protein expression. UVB radiation activated mitogen-activated protein kinases, phosphorylated (phospho)-extracellular signal-regulated kinase, phospho-c-Jun N-terminal kinase, and phospho-p38. FFO had a more cytoprotective effect than docosahexaenoic acid, the main component of fish oil, against UVB exposure-inducedcelldamage. Furthermore, the cytoprotective effectof FFO was evident in both UVB-exposed HaCaT cell and mouse models. Overall, these results demonstrate that FFO protects the skin against UVB-inducedoxidativestress through antioxidant effects. FFO may be developed as a preventive/therapeutic drug against UVB-induced skin damage.
Departments of 1 Advanced Convergence Technology & Science, 2 Veterinary Medicine, 3 Marine Life Sciences,
4 Civil and Enviromental Engineering, and 5 Nuclear and Energy Engineering, Jeju National University, Jeju 64243, Korea
(Received: January 22, 2016; Revised: March 13, 2016; Accepted: March 18, 2016)
Abstract : When exposed to gamma-rays, hair follicular cells immediately go through apoptosis, which hampers their rapid differentiation essential for the regeneration of hair. Phloroglucinol (PG) is a phenolic compound of Ecklonia cava, brown algae abundant in Jeju island, Korea. Containing plentiful polyphenols, PG is known for its instructive effects by inhibiting apoptosis, scavengingoxygen radicals, and protecting cells againstoxidativestress. In this study, we demonstrate that PG rescues radiosensitive hair follicular cells from gamma radiation-induced apoptosis and DNA damage. To identify protective capacity of PG on hair follicles, we irradiated with 8.5 Gy (1.5 Gy/min) of gamma- rays to the whole body of C57BL/6 mice at day 6 after depilation with or without PG. In mice exposed to radiation, the expression of proapoptotic molecule p53 was downregulated in the skin of PG treated group. On immunohistochemical observation of the skin, PG inhibited the immunoreactivity of p53 and cleaved caspase-3. PG treatment protected hair follicular cells from cell death due to gamma-radiation. Our results suggest that PG presents radioprotective effects by inhibiting apoptosis of radiosensitive hair follicular cells and can protect hair follicular cells from gamma-ray induceddamage.
et al., 2017). In addition, Samultang increased superoxide dismutase activity and xanthine oxidase inhibition activity without any toxic effect in mice model (Lee et al., 2010), supporting that Samultang may have the protective activity against free radical-induceddamage.
We further studied the protective effects of Samultang in a cellular system using SH-SY5Y cells. This cell is widely used as a model system of neuronal cell death induced by oxidativestress (Fordel et al., 2006). Therefore, the experimental model ofoxidativedamage in SH-SY5Y cell by free radicals may be useful for discovering effective protection ability from free radicals. To investigate the effects of Samultang on cell viability of H 2 O 2 - stressed SH-SY5Y neuronal cells, the cells were pre-treated with various concentration of Samultang (10, 50, and 100 μg/mL) and insulted with 300 μM H 2 O 2 . Fig. 1 showed the neuro-protective effects of Samultang on H 2 O 2 -induced SH-SY5Y cells under the MTT assay. The treatment of 300 μM H 2 O 2 diminished cell viability to 47.54% compared to 100% of the normal group. However, when cells were treated with Samultang, cell viability was signiﬁcantly elevated from the H 2 O 2 -induced neuronal cell death. In particular, the treatment with 50 μg/mL and 100 μg/mL of Samultang led to the increase ofcell viability up to 51.09% and 54.32%, respectively. The present results indicated that the Samultang had protective effects against H 2 O 2 -inducedoxidativestress in SH- SY5Y cells.
Hyung Don Kim, Eun Young Lee, Jeong-Yong Park, Kyung Hye Seo, Kang-Hyo Lee, Jehun Choi, Jae-Gu Han, Jae-Han Cho*, and Seung Eun Lee*
Department of Herbal Crop Research, NIHHS, RDA, Eumseong, Chungbuk 27709, Korea.
ABSTRACT: This study was carried out to identify medicinal mushrooms with protective effects againstoxidativestress in PC12 neuronal cell line, followed by evaluation of their antioxidant property. Extracts of medicinal mushrooms, including Ganoderma lucidum extract (GLE), antler-shaped Ganoderma lingzhi extract (AGLE), Hericium erinaceus extract (HEE), and Sanghuangporus baumii extract (SBE), were screened for cytotoxicity using MTT assay. None of the extracts up to 10 µg/ml concentration affected cell viability. These extracts were further checked for their protectiveeffectagainstoxidativestress-inducedreactiveoxygenspecies (ROS) production. Exposure to 50 µM H 2 O 2 induced ROS generation in PC12 cells, which was inhibited only by treatment with AGLE. In addition, inhibition of H 2 O 2 -induced ROS generation by AGLE was found to be in a dose-dependent manner (2.5, 5, and 10 µg/ml). Microscopic examination of DCF fluorescence for detection of ROS showed a similar pattern.
〔핸 eJ 띤 rnat01Medicineand Life SCience ~!:J 한쁘， Jic 깐앤〕
GaJangin protects hydrogen peroxide inducedoxidativestressvia the scavengingof
- - reactlve oxygen speCles
Mei .Jing Piao ， Kyoung Ah Kang ， and .Jin Won Hyun Departmenl018iochemistry ， Jeju NationalUniversitySCh 。이 이 Medicine.Jeju ， Korea
L 맨턴쁘띤힌 이 M명i 히낀 e and 민 e 혼 ience V 。 I~ ~， N~~~ ~.Q 영」
Arltioxidant effects of geraniin viascavengingreactiveoxygenspecies
Dong Ok Ko ， Kyoung Ah Kang ， and'Jin Won Hyun
Departmenl 이 Biochemistry ‘ Jeju Nalional Un;versllySchool 이 Medicine ‘ Jeju. Korea
3-3. Baicalein protects DNA, lipids, and proteins against UVB-inducedoxidativedamage
We next investigated whether baicalein can inhibit damage to macromolecules in UVB- exposed cells. First, we monitored UVB-induced DNA damage using the comet assay. The length of comet tails in microscopy images and the percentage of cellular fluorescence in tails are shown in Figure 3A. After treatment of cells with UVB radiation, the comet tail length was distinctly elongated, as well as the ratio of damaged DNA outside of nuclei.
cell의 산화적 스트레스 개선효과가 뛰어나 활성 획분층 으로 사료된다.
IV. 결 론
본 연구에서는 broccoli flower와 broccoli sprout의 MeOH 추출물을 CH 2 Cl 2 , BuOH 및 H 2 O 획분으로 분획하여 각 획분층의 라디칼 소거능과 LLC-PK 1 cell을 이용한 산화 적 스트레스 개선효과를 살펴보았다. LLC-PK 1 cell을 이 용한 실험에서 broccoli sprout가 broccoli flower에 비해 AAPH에 의한 산화적 스트레스 개선 효과 우수하였으며, 각 획분중 BuOH 획분이 가장 뛰어난 효과를 보여, broccoli flower와 broccoli sprout의 BuOH 획분을 50 μg/mL로 처리하였을 때 각각 83.5%와 85.1%의 세포생존율을 나타 내었다. 또한 broccoli sprout의 추출물 및 분획물의 DPPH 라디칼 소거능이 우수하였으며, 특히 BuOH 획분의 경우 19.32 µg/mL의 IC 50 값을 나타내었다. 이상의 결과로부터 broccoli flower에 비해 broccoli sprout의 항산화활성이 우수함을 알 수 있었으며 각 획분중 BuOH 획분이 라디칼 소거능과 peroxyl radical에 의한 산화적 스트레스 개선효 과가 가장 우수하여 활성 획분층으로 사료된다.
However, although miconazole induced LC3‑II expression,
Figure 4. ROS‑ and ER stress‑inducing effects of miconazole in relation to autophagic cell death. (A) ROS production in cells treated with miconazole for 10 min, as measured by flow cytometry. Fluorescence intensity is directly proportional to the quantity of ROS species in the cells. (B) Levels of LC3‑I and LC3‑II proteins in U251MG cells treated with miconazole in the presence or absence of 5 mM NAC, as determined by western blotting and densitometry using β ‑actin as a loading control. (C) Levels of BiP, IRE1 α and CHOP proteins in cells pretreated with 5 mM NAC for 2 h and then treated with miconazole at the indicated concentrations for 24 h, as determined by western blotting using β ‑actin as a loading control. Densitometric analysis of the western blots is shown in the bar graph. Densitometric and cell viability data are the mean ± standard deviation of three independent experiments. * P<0.05, ** P<0.01 and
본 연구에 활용된 香砂平胃散은 平胃散을 基本方으로 하여 香附子, 木香, 砂仁을 비롯 한 다섯 가지 藥物을 加味한 處方으로 萬病 回春 7) 에 최초로 수록되어, 傷食, 食鬱證, 食 積泄, 停食嘔吐, 呑酸吐酸, 嘈雜, 瘀血 및 食 積으로 인한 腹痛, 食積 脇痛 등의 치료에 사용된다 하였다. 또한 韓方 臨床에서는 平 胃散, 香砂養胃湯과 더불어 心痛의 치료에도 활용되고 있다. 한편 香砂平胃散은 ICR계 생쥐에 이식된 sarcoma-180 종양세포의 성 장을 억제하고 자연살해세포(NK cell)와 림 프구의 활성을 높임으로써 항암 및 면역증 강 효능을 나타낸다고 보고된 바 있다 8) . 그
WI-38 cell에서의 H 2 O 2 에 의한 산화적 스트레스에 대해 우수한 세포 보호효과를 보였다. 가지의 과피의 주된 색소 인 delphinidin는 DPPH 소거능을 측정한 결과, IC 50 가 6.59 µg/mL로 아주 강한 DPPH 소거능을 보였다. 또한 AAPH에 의한 LLC-PK 1 cell의 산화적 스트레스에 대해 세 포생존율을 증가시킴으로써 delphinidin의 세포 보호효과 가 아주 뛰어난 것을 확인할 수 있었으므로, 우수한 항산화 제로서의 활성을 나타낼 것으로 사료된다.
결과 및 고찰
총 페놀화합물 함량
플라보노이드류, 페놀산류 및 식물성 에스트로겐류를 포함하는 총 페놀화합물은 식물의 2차 대사산물로, 페놀릭 하이드록실기를 2 개 이상 가지고 있다. 이를 통한 수소공여로 라디칼과 반응하여 공명구조를 통해 안정화됨으로써 산화를 방지하는 활성 및 항노 화 등과 같은 생리효과를 가지게 된다(4). 이에 와송의 산화방지 효과를 알아보기 위한 실험의 시작점으로 와송의 분획물별(노말 헥세인, 클로로폼, 아세트산에틸 및 증류수) 총 페놀화합물 함량 을 측정하였으며, 결과는 다음과 같다(Fig. 2). 각각의 분획물의 총 페놀화합물 함량은 99.00, 129.83, 222.92 및 81.58 mg GAE/g of dried Orostachys japonicus A. Berger 으로, 아세트산에틸 분획 물(EFOJ)에서 가장 우수한 함량을 나타냈다. 이는 분획을 통해 와송에 함유되어 있는 페놀성 성분들이 극성도에 따라 용매별로 분획된 것으로 판단된다. 총 페놀 화합물은 ABTS, DPPH 라디 칼 소거 활성, 환원력 및 과산화 지방질 억제와 같은 산화방지 활성에 중요한 인자로 작용한다(10). 이에 따라 가장 높은 총 페 놀화합물 함량을 나타낸 아세트산에틸 분획물(EFOJ)을 이용하여 이후 실험을 진행하였다.
게다가 V79-4 chinese hamster lung fibroblast cell에서 H 2 O 2 처리시 세포 내 ROS가 상당히 증가함을 확인할 수 있었다. Luteolin처리시 H 2 O 2 로 유도된 세포 내 ROS와 cellular components인 lipid와 DNA손상을 줄임을 확인하였다.
Luteolin처리시 세포 생존률을 증가시켰으며, H 2 O 2 로 유도된 apoptosis를 mitochondria mediated caspases pathway를 통해 억제함을 나타내었다. Luteolin은 active caspase 3, 9 그리고 Bax의 발현을 줄였고, bcl-2의 발현을 증가시켰다.
Melatonin, an antioxidant synthesized in the pineal gland, plays multiple roles in various physiological mechanisms. Melatonin exerts neuroprotective effects in the central nervous system. To determine the effectof melatonin on NSCs which is in LPS-induced inflammatory stress state, we first investigated nitric oxide (NO) production and cytotoxicity using Griess reagent assays, LDH assay, and neurosphere counting. Also, we investigated the effectof melatonin on NSCs by measuring the mRNA levels of SOX2, TLX, and FGFR-2. In addition, western blot analyses were performed to examine the activation of PI3K/Akt/Nrf2 signaling in LPS- treated NSCs. In the present study, we suggested that melatonin inhibits NO production and protects NSCs against LPS-induced inflammatory stress. In addition, melatonin promoted the expression of SOX2 and activated the PI3K/Akt/Nrf2 signaling under LPS-induced inflammation condition. Based on our results, we conclude that melatonin may be an important factor for the survival and proliferation of NSCs in neuroinflammatory diseases.