골풀에서 분리한 Dehydroeffusol의 세포보호 효과와 그 메커니즘에 대한 연구

원두현 서울과학기술대학교 2013 국내석사

이번 연구의 목적은 기존에 광증감 반응이 야기하는 세포 손상에 대해 높은 방어 효과를 나타낸다고 알려져 있던 골풀 고갱이 부위의 추출물의 원인 물질을 찾고 그 방어 메커니즘을 규명하는 것이었다. 우선 골풀 고갱이 부위에서 높은 세포 보호 효과의 원인 물질을 분리 및 구조 분석한 결과 “Dehydroeffusol”인 것을 확인하였다. 메커니즘 실험을 진행하기에 앞서 L-ascorbic acid, α-tocopherol, phenanthrene을 비교 물질로 선정하였고 광용혈법을 이용해 세포 보호 효과를 확인하였다. 세포 보호 효과는 dehydroeffusol > α-tocopherol으로 나타났고, L-ascorbic acid와 phenanthrene은 세포를 보호하지 못하는 것으로 나타났다. Dehydroefusol이 나타내는 높은 세포 보호 효과의 메커니즘을 밝히기 위해 singlet oxygen 소광 속도 상수, 자유 라디칼 소거 활성, 다양한 ROS에 대한 소거 활성 실험을 진행하였다. 여기에 더하여 이러한 항산화 요인이 효과를 나타내는데 영향을 미칠 수 있는 적혈구 세포 침투율을 확인해 보았다. 데이터를 종합적으로 분석한 결과, L-ascorbic acid의 실험 결과를 통해 항산화 능력이 뛰어나도 세포막 내부로 침투하지 못하면 세포를 보호할 수 없다는 것을 확인할 수 있었다. 또한 phenanthrene을 통해 세포 침투율이 높아도 항산화 능력을 갖지 못하면 세포 보호 효과를 나타내지 못하는 것으로 확인하였다. Dehydroeffusol과 α-tocopherol의 데이터를 비교한 결과, 다양한 ROS 소거 능력이 다른 항산화 효과에 비해 세포를 보호하는데 중요한 작용을 하는 것으로 확인하였다. 추가적으로 dehydroeffusol의 높은 ROS 소거 능력이 킬레이트 반응이 아닌 항산화 능력에 기인한 것으로 확인하였다. 따라서 이번 연구를 통해 Type II 반응에서 부가적으로 생성되는 ROS가 광증감 반응이 야기하는 세포 용혈의 주요 원인이고, 이러한 원인으로부터 세포를 보호하는 작용에서 항산화 물질의 세포 침투와 다양한 ROS 소거 능력이 큰 영향을 미치는 것으로 확인하였다. Previous study reportred that extract from medulla part of Juncus effusus L. has prominent cellular protective effect on photosensitization reaction. Purpose of this study is finding the substance responsible for high cellular protective effect of medulla part of Juncus effusus L. and investigating the cell protection mechanism. A single substance which shows cellular protective effect was isolated from the extract of medulla part of Juncus effusus L. and identified as “Dehydroeffusol”. Before the investigation of mechanism, L-ascorbic acid, α-tocopherol, phenanthrene were selected as references and cellular protective effect was tested using photosensitized hemolysis of human erythrocytes. Dehydroeffusol showed higher cellular protective effect than α-tocopherol. On the other hand, cellular protective effects were not observed in L-Ascorbic acid and phenanthrene. To investigate the mechanism of prominent cellular protective effect of dehydroeffusol, singlet oxygen quenching rate constant, free radical scavenging activitiy, reactive oxygen species (ROS) scavenging activity on various ROS produced by Type II photosensitization reaction were tested to check the antioxidative factors related to photosensitization reaction. Additionally, erythrocyte membrane penetration efficiency which can affect functions of the antioxidative factors was checked. From the comprehensive anlaysis of data, the results of L-ascorbic acid confirm that if there is no cell membrane penetration, there is no cellular protective effect despite high antioxidative effect. Besides, the results of phenanthrene confirm that antioxidative activity is requirement for cell protection. By comparison of dehydroeffusol and α-tocopherol, various ROS scavenging acitivity is more important factor for protecting cell than other antioxidative activities. Additionally, by iron chelation assay, it is checked that various ROS scavenging activity of dehydroeffusol is not because of chelation effect, but because of its antioxidative ability. These results demonstrate that incidentally produced ROS from Type II reaction is main reason for cell hemolysis by photosensitization reaction, and cell membrane penetration and various ROS scavenging ability have an important effect on cell protection.

Antiulcer Activity, Toxicity, and Possible Mechanisms of Action of Opuntia ficus-indica Fruit-Derived Materials

박상욱 서울대학교 대학원 2021 국내박사

Gastritis is a common disease among Korean adults who take mainly very salty and spicy foods. Usually, symptoms include epigastric pain, nausea, vomiting, abdominal pain, indigestion, and bloating. Pathophysiology of gastritis is due to a lack of equilibrium between the gastric aggressive factor (acid, pepsin, and Helicobacter pylori) and the mucosal defense factor (gastric mucus, bicarbonate secretion, prostaglandins, and innate resistance of the mucosal cells). There are several types of medicines used to treat a gastric ulcer. However, these treatments have side effects. There is a pressing need to develop a new gastritis treatment with fewer side effects. Plants are regarded to represent a reservoir of potential therapeutics and therefore the efforts to search for novel compounds from medicinal plants have been continued. Opuntia ficus-indica (Cactaceae) has been used in traditional medicine of many countries. It is widely cultivated in Jeju Island, Korea, for use in the manufacture of health foods. The aim of this study was to develop O. ficus-indica fruits extract as an antiulcer botanical drug. The antiulcer activity of OF-80E (80% ethanol extract of O. ficus-indica fruits) was assessed using the ethanol-, non-steroidal anti-inflammatory drugs (indomethacin, aspirin, and diclofenac)-, and stress-induced gastritis rat models. The results were compared with those of commercially available drugs, Stillen® tablet and Mucosta® tablet. In addition, the acute toxicity, sub-chronic toxicity, genotoxicity, and safety pharmacology studies of OF-80E were analyzed under Organization for Economic Cooperation and Development guideline and Good Laboratory Practice regulations for human safe consumption. Finally, the possible mechanism underlying the antiulcer actions of OF-80E in gastritis models were elucidated using biochemical and molecular analyses. Inhibition of aspirin-induced cytotoxicity in AGS cells assay-guided fractionation of the O. ficus-indica fruits led to the identification of two active compounds through spectroscopic analyses, including electron ionization mass spectrometry and nuclear magnetic resonance spectroscopy. The two antiulcer constituents were the flavonoids aromadendrin and narcissin. Based on the IC50 values, the flavone, aromadendrin (<0.5 μM), and the flavonol, narcissin (<0.5 μM), were more effective in inhibition of aspirin-induced cytotoxicity in AGS cells than other flavones, naringenin (5.9 μM), eriodictyol (>10 μM) and taxifolin (1.1 μM), and flavonols, kaempferol, quercetin, and isokaempfride (>10 μM). OF-80E was more effective than commercially available drugs to protect gastric mucosal damage against aggressive factors. In ethanol-, non-steroidal anti-inflammatory drugs-, and stress-induced gastritis rat models, OF-80E inhibited gastric hemorrhagic lesions and histological tissue damage effectively comparing than commercially available drugs. In a single dose oral toxicity study, the approximate lethal dose of OF-80E in both male and female of Sprague Dawley (SD) rats was higher than 10000 mg/kg. In a 13-week repeated oral toxicity study, the no observed adverse effect level of OF-80E was 2000 mg/kg/day for both sexes of SD rats. In a 4-week repeated oral toxicity study, the maximum tolerance dose of OF-80E was 1500 mg/kg/day for both sexes of beagle dogs. In Salmonella typhimurium and Escherichia coli reverse mutation studies, OF-80E did not cause mutation. In a chromosome aberration test, OF-80E did not cause chromosomal aberration in Chinese hamster lung cells. In micronucleus assay, OF-80E did not induce micronuclei in the mammalian bone marrow cells. Single oral administration of OF-80E to rodent at below 5000 mg/kg did not affect the central nervous system of ICR mice and did not induce adverse effects on the respiratory system of SD rats. OF-80E did not effect on the human ether-a-go-go related gene channel up to the concentration of 500 μg/mL, indicating that the effect of OF-80E on cardiovascular system was to be low. OF-80E increased glutathione reduced by aspirin in AGS cells and decreased by indomethacin in rats. OF-80E increased prostaglandin E2 levels reduced by aspirin in AGS cells. Decreased adherent mucus was synthesized and stimulated, by OF-80E pretreatment in indomethacin-induced gastritis rats. OF-80E inhibited myeloperoxidase activity in indomethacin-induced rat gastric mucosal and reduced tumor necrosis factor-α in stress-induced rat gastric mucosal. 위염은 주로 짠 음식과 매운 음식을 주로 먹는 한국 성인들에게 흔한 질병이 다. 일반적으로 증상에는 상복부 통증, 메스꺼움, 구토, 복통, 소화 불량 및 복부 팽 만감이 있다. 위염은 병태생리학적으로 위 공격 인자 (산, 펩신, 헬리코박터 파일로 리)와 위 점막 방어 인자 (위 점액, 중탄산염 분비, 프로스타글란딘 및 점막 세포의 선천적인 저항성) 사이의 불균형으로 발병한다. 위궤양 치료제로 여러 약물이 사용 되고 있다. 그러나 이러한 치료제에는 부작용이 있다. 부작용이 적은 새로운 위염 치료제 개발이 절실히 요구되고 있다. 식물은 잠재적인 치료제를 가지고 있을 것으 로 생각되어 약용 식물에서 새로운 화합물을 찾기 위한 노력이 계속되고 있다. Opuntia ficus-indica (선인장과) (백년초)는 많은 국가에서 전통 의약품으로 사용 되고 있다. 대한민국의 제주도에서는 건강 식품 제조 용도로 널리 재배되고 있다. 본 연구는 백년초 열매 추출물을 위염 치료 천연물의약품으로 개발하기 위해 수행 되었다. OF-80E (백년초 열매 80% 에탄올 추출물)의 항궤양 활성은 에탄올, 비스테로 이드성 항염증제 (인도메타신, 아스피린 및 디클로페낙) 및 스트레스 유발 위염 랫 트 모델을 사용하여 평가하였다. 그 결과는, 시판된 약물인 스티렌정 (Stillen® tablet) 및 뮤코스타정 (Mucosta® tablet)과 비교하였다. 또한, OF-80E의 급성독성, 아만성독성, 유전독성 및 안전성 약리 연구는 인간의 안전한 섭취를 위해 경제협력 개발기구 지 침 및 우수실험실관리 규정에 따라 평가하였다. 마지막으로, 위염 모델에서 OF-80E 의 항궤양작용의 가능한 메커니즘은 생화학 및 분자 분석을 사용하여 설명하였다. AGS 세포를 이용한 아스피린 유도 세포독성 억제 시험을 기반으로 성분 분리 를 통하여 백년초 열매로부터 분리하고, 전자이온화 질량분석법과 핵자기 공명 분 광법을 포함한 분광 분석을 통해 두 종의 활성 화합물을 동정하였다. 두 종의 항궤 양 성분은 플라보노이드인 aromadendrin과 narcissin으로 확인되었다. IC50 값으로 보 면, flavone인 aromadendrin (<0.5 μM)과 flavonol인 narcissin (<0.5 μM)은 다른 flavones인 naringenin (5.9 μM), eriodictyol (>10 μM), taxifolin (1.1 μM) 및 다른 flavonols인 kaempferol, quercetin 및 isokaempfride (>10 μM) 보다 AGS 세포를 이용한 아스피린 유도 세포독 성 억제 효과가 우수하였다. OF-80E는 시판된 약물보다 공격인자에 대해서 위 점막 손상을 보호하는 효과 가 우수하였다. 에탄올, 비스테로이드성 항염증제 및 스트레스 유발 위염 랫트 모델 에서, OF-80E는 시판된 약물에 비해 효과적으로 위 출혈성 병변과 조직학적 조직 손상을 억제하였다. 단회 경구투여 독성연구에서 OF-80E의 개략적인 치사량은 SD 랫트의 암, 수 모두에서 10000 mg/kg 이상으로 확인되었다. 13주 반복 경구투여 독성연구에서 OF- 80E의 무독성량은 SD 랫트의 암, 수 모두에서 2000 mg/kg/day로 확인되었다. 4주 반 복 경구투여 독성 연구에서 OF-80E의 최대내성용량은 비글견 암, 수 모두에서 1500 mg/kg/day로 확인되었다. Salmonella typhimurium과 Escherichia coli를 이용한 복귀돌연 변이 시험에서 OF-80E는 돌연변이를 유발하지 않았다. Chinese hamster lung 세포를 이용한 염색체 이상시험에서 OF-80E는 염색체 이상을 유발하지 않았다. 소핵시험에 서 OF-80E는 동물 골수세포에서 소핵을 유발하지 않았다. OF-80E를 5000 mg/kg 이하 로 설치류에 단회 경구 투여했을 때, ICR 마우스의 중추신경계에 영향을 미치지 않 았고, SD 랫트의 호흡기계에 영향을 미치지 않았다. OF-80E는 500 μg/mL 농도까지 human ether-a-go-go related gene 채널에 영향을 미치지 않는 것으로 보아, OF-80E는 심 혈관계에 미치는 영향이 낮을 것으로 확인되었다. OF-80E는 AGS 세포에서 아스피린에 의해 감소되고, 랫트에서 인도메타신에 의해 감소한 glutathione을 증가시켰다. OF-80E는 AGS 세포에서 아스피린에 의해 감 소한 prostaglandin E2의 농도를 증가시켰다. 인도메타신 유도 위염 랫트에서 감소된 부착성 위점액은 OF-80E의 처리에 의해 합성되고 분비되었다. OF-80E는 인도메타신 유도 랫트의 위 점막에서 myeloperoxidase의 활성을 억제하고, 스트레스 유도 위염 랫트의 위 점막에서 tumor necrosis factor-α를 감소시켰다.

Detection of the Protective Effect of Black Rice Using the Renal Toxicity Model

Kwon, Hye Ri Graduate School, Eulji University 2018 국내석사

Vancomycin hydrochloride (VAN), which is used as an antibiotic, causes side effects of nephrotoxicity. It was showed renal excretion abnormalities, tubule damage, and inflammatory responses. Black rice (BR) is a kind of rice, and BR contains abundant anthocyanin which has antioxidant, anti-cancer and anti-ulcer effect. The purpose of this study is to evaluate the protective effect of BR against VAN-induced nephrotoxicity. Five weeks male Sprague-Dawley rats were randomly divided into control (CON) group, VAN group and BR group. The BR group was orally administered 10 ml/kg of BR extract once a day for 14 days. During the same period, the CON and VAN groups were orally administered 10 ml/kg of distilled water (D.W). The VAN and BR groups were intraperitoneally (i.p) injected 400 mg/kg of VAN once a day for the last 3 days after 1hr of administration of treatment materials. During the same period, the CON group was intraperitoneally (i.p) injected 5ml/kg of saline after administration of D.W. After 24 hr of last VAN injection, rats were sacrificed. Serum blood urea nitrogen (BUN) and creatinine levels decreased in the BR group compared to VAN group. In the renal cortex of BR group, the degree of damage such as tubular dilatation and epithelial cell desquamation decreased compared to VAN group. Glomerulus and tubules basement membrane in the BR group were regular and clear compared to VAN group. The expression of Tumor necrosis factor-alpha (TNF-α) and N-acetyl-D-Glucosaminidase (NAG) decreased in the BR group compared to VAN group. As a result of Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) stain, the number of positive cells in the BR group decreased compared to VAN group. Tissue malondialdehyde level decreased in the BR group compared to VAN group. In conclusion, the data of this experiment showed that the BR extract had protective effects by antioxidant, anti-inflammatory and anti-apoptotic against VAN induced nephrotoxicity. 항생물질로 사용되고 있는 반코마이신은 신독성이라는 부작용을 일으킨다. 반코마이신으로 인한 신독성으로 인해 신장 배설 기능의 이상, 세뇨관 손상, 염증 반응이 나타난다. 흑미는 벼의 한 종류이고, 흑미에는 항산화, 항암, 항궤양 효과가 있는 안토시아닌이 풍부하게 함유되어있다. 본 연구의 목적은 반코마이신으로 인한 신독성에 길항하는 흑미의 보호효과에 대하여 평가하는 것이다. 5주령 Sprague-Dawley 랫드 수컷을 무작위로 대조군(CON), 반코마이신군(VAN), 흑미군(BR)으로 나누었다. 흑미군은 2주 동안 10 ml/kg의 흑미 추출물을 경구투여 하였다. 같은 기간 동안, 대조군 그리고 반코마이신군은 10 ml/kg의 증류수를 경구투여 하였다. 반코마이신, 흑미군은 마지막 3일 동안 하루에 한번, treatment 물질 투여 1시간 후 400 mg/kg의 반코마이신을 복강내주사 하였다. 같은 기간 동안, 대조군은 증류수 투여 1시간 후에 5 ml/kg의 생리식염수를 복강내주사 하였다. 마지막 반코마이신 투여 하루 뒤 랫드를 희생하였고, 생화학적 분석을 위해 blood sample을 수집하였다. 신장은 조직학적 평가를 위해 제거하였다. 흑미군에서 혈청 blood urea nitrogen과 creatinine 수치가 반코마이신군과 비교하여 감소하였다. 흑미군의 신장 겉질에서, 세뇨관 확장, 상피세포 박리, 내강 내 cast가 반코마이신군과 비교하여 감소하였다. 흑미군에서 사구체와 세뇨관의 기저막은 반코마이신군과 비교하여 규칙적이고 명확했다. Tumor necrosis factor-alpha 와 N-acetyl-D-Glucosaminidase 발현은 반코마이신군과 비교하여 흑미군에서 감소하였다. Terminal deoxynucleotidyl transferase dUTP nick end labelling 염색 결과, 흑미군에서 positive cell의 수가 반코마이신군과 비교하여 감소하였다. 또한, 흑미군에서, 조직 malondialdehyde 수치는 반코마이신군과 비교하여 감소하였다. 결론적으로, 본 실험의 데이터는 흑미 추출물이 반코마이신으로 인한 신독성에 길항하여 항산화, 항 염증, 항 세포자멸사의 보호효과를 가진다는 것을 보여주었다.

Chemoprotective effects of sodium citrate on genotoxic and non-genotoxic outcomes in rats treated with carcinogens and metals

임성광 성균관대학교 일반대학원 2017 국내박사

Benzo[a]pyrene (B[a]P), N-Nitrosodimethylamine (NDMA) and toxic metals are well-known toxicants, which are easily exposed in the surroundings including tobacco, polluted air, ingestion of contaminated food and water. These toxicants cause mayy types of hazard responses such as mutagenesis, carcinogenesis, reproductive toxicity, oxidative stress and inflammatory response. Citrate, a generally recognized as safe (GRAS) substance, is used and is ingested frequently in the surroundings including foods. Citrate does not produce mutagenic, reproductive or developmetal toxicities. Citrate has also beneficial effects including antioxidant and anti-inflammatory effects. However, few studies have been reported on the anti-mutagenic effect of citrate. The present study investigates the protective effects of citrate on the carcinogens- and metals-induced toxicities such as mutagenesis, oxidative stress, inflammation and reproductive toxicity. For first study, B[a]P (10 mg/kg body weight) and NDMA (1 mg/kg body weight) was administered to rats in corn oil via oral gavage over a 28 days period. Sodium citrate (258, 516, and 1032 mg/kg) was co-administered with B[a]P and NDMA for 28 days. Expression levels of CYP1A1 and CYP2E1 increased in the liver of rats treated with B[a]P and NDMA decreased in a dose-dependent manner by co-treatment with sodium citrate. Sodium citrate reduced the BPDE-DNA adduct formation in liver as well as NDMA-DNA adduct. Sodium citrate recovered the changes of oxidative and inflammatory marker and serum testosterone by B[a]P and NDMA. And antioxidant and anti-inflammation effects of sodium citrate were identified. For second study, aluminum chloride (33.11 mg/kg), cadmium chloride (0.88 mg/kg), lead(II) acetate trihydrate (46.65 mg/kg), methylmercury chloride (0.299 mg/kg), nickel(II) chloride (1.05 mg/kg) and sodium arsenite (0.41 mg/kg) was administered to rats in distilled water via oral gavage over a 28 days period. Sodium citrate (258, 516, and 1032 mg/kg) was co-administered with metals for 28 days. Sodium citrate showed the protective effects on the oxidative stress and hormone imbalance by metals although the effects are not large. So Our results also showed that sodium citrate has the protective effect on the oxidative, inflammatory, reproductive and carcinogenic toxicities by B[a]P, NDMA and metals in rats.

The Effects of The 57-kDa Protein Isolated from Korean Royal jelly on the Activity of Human-derived Osteoblast : 국산 로얄젤리(royal jelly)에서 분리한 57-kDa 단백질의 인간유래조골세포 활성에 미치는 영향

최새움 서울대학교 대학원 2014 국내석사

ABSTRACT Royal jelly (RJ) is a secretion product of the cephalic glands of nurse bees that has been used for centuries for its extraordinary properties and health effects. Pharmacological actions of RJ such as antitumor, antiviral, and antioxidant activity have been well noted. RJ and its 57-kDa protein were also reported to enhance the bone-forming ability of osteoblasts and the production of albumin and to have protective effect on rat liver cells. However, the impact of RJ -derived materials, particularly 57-kDa protein, on Saos-2, a human osteoblast-like cell line, is not yet fully understood. Also, no information has been done to consider potential use of RJ -derived materials to manage ultra violet (UV) induced damages. In this study, the effects of RJ and its 57-kDa protein on Saos-2 and the cytoprotective activity of the materials toward Saos-2 cells damaged by UV were evaluated. The cytoprotective activity of the materials was compared with that of N-acetyl-L-cysteine (NAC), a currently used antioxidant, for use as future commercial products with cytoprotective action using a MTS assay. Firstly, 57-kDa protein was purified from domestic RJ using fast protein liquid chromatography and gel filtration method. The 57-kDa protein at concentrations between 10 and 100 μg/mL exhibited a high correlation between protein concentration and cell viability (correlation coefficient (R2) > 0.85). Pretreatment with 0.1 mg/mL 57-kDa protein and 2 mM NAC to Saos-2 cells resulted in 79 and 77% protection against UVB, respectively, whereas 1.5 mg/mL RJ showed no statistically significant protection from radiation induced cell death. Osteoblast extracellular Ca2+-sensing receptor regulates bone development, mineralization, and turnover (Dvorak-Ewell MM et al. 2011). The mitochondria are to generate the cell energy, ATP (i.e., phosphorylation of ADP), and Changes in alkaline phosphatase level and activity are involved in a variety of physiological and pathological events, such as bone development. The effects of 57-kDa protein on the calcium sensing receptor in live cells and mitochondria were compared with cell groups pretreated with UV using confocal microscopy. The microscopic observation revealed that the cells treated with 57-kDa protein showed normal cell shapes and organelles being maintained, similar to normal cells. Pretreatment of Saos-2 cells with 0.1 mg/mL 57-kDa protein significantly resulted in the increase in ALP activity from radiation induced cells. However, 1.5 mg/mL RJ and 2 mM NAC exhibited no statistically significant increase in ALP activity. In conclusion, 57-kDa protein can be useful for the bone formation of osteoblasts and for protection from UV. For practical use of RJ and 57-kDa protein as novel anti- UV induced stress products to proceed, further research is needed to establish their human safety and whether this activity could be exerted in vivo after consumption of the product by humans. In addition, their anti-UV induced stress modes of action need to be established and detailed tests are needed to understand how to improve anti-UV induced stress potency and stability for eventual commercial development.

(The) effects of news about flu on the persuasiveness of hand washing campaign and product advertising messages : an application of priming theory and protection motivation theory

령홍심 Graduate School, Korea University 2012 국내석사

This thesis investigated how news about flu can influence the persuasiveness of hand-washing campaign and product advertising messages. Based on priming theory, it was expected that priming flu in the news would increase audiences’ attitudes toward the campaign and ad messages as well as their intentions to engage in hand washing behaviors and product purchase behavioral intentions. In addition, based on protection motivation theory, this effect was expected to be mediated by perceived severity. 108 Malaysia students from UCSI University participated in the experiment, and they were randomly assigned to the priming or non-priming conditions. Participants in the priming condition read a news message and then viewed a campaign message as well as an ad message. On the other hand, participants in the non-priming condition viewed the same campaign and ad messages without reading the news message. Results suggest that news priming affects campaign attitudes and ad product purchase intentions. As expected, compared with the non-priming condition, respondents in the priming condition showed more positive attitudes toward the campaign message and greater intention to use the advertised product. The effect was mediated by perceived severity. This research has practical and theoretical implications. Because news priming affects campaign attitudes and ad product intentions, health campaign and ad practitioners may want to increase campaign expenditures when the issue has been made salient by the news media. In theoretical terms, this research attempted to connect priming theory and protection motivation theory to understand the joint effects of news, health campaign, and advertising messages.

Protective effect of extracellular vesicles released from the neural stem cells on 6-hydroxydopamine induced neurotoxicity

이은지 서울대학교 대학원 2018 국내석사

Neural stem cells (NSCs) are potential therapeutic resources for Parkinson’s disease (PD) because they promote recovery of neuronal damage. NSC-secreted extracellular vesicles (EVs) are key mediators of positive paracrine effects. Direct evidence for neuronal protective effects of EVs is essential for developing new PD therapeutics. In this study, we observed the protective effects of NSC-derived EVs during neurotoxin 6-hydroxydopamine (6-OHDA)-induced degeneration of SH-SY5Y dopaminergic cells. To trace EV movement, a lentivirus containing Palm tandem dimer tdTomato (Palm-td) was transduced into F3 NSCs. EVs isolated from Palm-td–infected F3 cells showed high tdTomato fluorescence intensity. We found that pre-treatment with EVs dramatically prevented 6-OHDA-induced toxicity by reducing intracellular reactive oxygen species (ROS), percentage of apoptotic cells, and caspase-3/7 activity. These results indicate that NSC-derived EVs have neuroprotective effects against 6-OHDA-induced cell damage, possibly through anti-oxidant and anti-apoptotic action. The findings may provide further insights into potential therapeutic strategies using NSC-derived EVs for neuronal protection against oxidative stress.

Application of Herb Ingredient and Subunit Vaccine as New Strategies for Controlling Brucellosis

트란쉬안응옥후이 경상대학교 대학원 2020 국내박사

Brucellosis is a common zoonotic and contagious disease caused by genus Brucella. Brucella infections have resulted in devastating losses to livestock industry as well as significant burdens to human healthcare system in developed and developing countries. Therefore, many control and eradication strategies against animal and human brucellosis have been implemented in the last few decades. Due to some drawbacks of the use of antibiotics in brucellosis treatment such as restricted choice of intracellular antibiotics, prolonged treatment, relapse, antibiotic resistance, two or more antibiotics combination and overlap between treatment regiments of two diseases, vaccination and herbal medicine have been considered as promising alternative therapies to antibiotics. The first chapter of this study investigated the protective efficacy of Emodin, an active, naturally-occurring anthraquinone derivative of several traditional Chinese herbs, against Brucella abortus (B. abortus) infection in macrophages. Brucella was incubated with different concentrations of Emodin and showed that bacterial survival rates were markedly reduced in a dose-dependent manner at increasing incubation time points. Through bacterial infection assay, the highest non-cytotoxic concentration of Emodin demonstrated attenuated invasion of Brucella into macrophages, however it did not inhibit the growth of this pathogen within the host cells. On the other hand, Emodin effectively decreased the number of bacteria that adhered to host cells, which indicated its potential as an anti-adhesion agent. Furthermore, using immunoblotting and fluorescence-activated cell sorting (FACS) assay for detecting mitogen-activated protein kinase (MAPK) signaling proteins and F-actin polymerization, respectively, the results showed that the Emodin-incubated cells displayed modest reduction in the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and inhibition of F-actin polymerization as compared to control cells. These findings indicate the potential use of Emodin as a naturally-occurring alternative method for the prevention of animal brucellosis. The second chapter of this study investigated the protective effects of two recombinant B. abortus proteins adenylate kinase (Adk) and preprotein translocase subunit (SecB) against Brucella infection in a mouse model. Two recombinant proteins encoded by B. abortus genes Adk and SecB were evaluated as single subunit vaccine (SSV) as well as combined subunit vaccine (CSV2) against B. abortus infection in BALB/c mice. These genes were cloned into pcold-TF expression system and recombinant proteins were expressed in Escherichia coli (E. coli) DH5α. The immunoreactivity of purified rAdk and rSecB was analysed by immunoblotting showing that purified rAdk and rSecB as well as pcold-TF vector strongly reacted with Brucella-positive serum. Mice were immunized intraperitoneally with SSVs, CSV2, pcold-TF, RB51 and phosphate-buffered saline (PBS). The analysis of cytokine revealed that SSVs and CSV2 can strongly induce production of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), suggesting that these subunit vaccines elicited innate immune response, particularly, activated antimicrobial mechanism of macrophages to limit the initial infection. On the other hand, immunization with SSVs and CSV2 elicited strong interferon gamma (IFN-γ) production and decreased interleukin 10 (IL-10) production compared to PBS group. The secretion profiles of IFN-γ and IL-10 together with an enhancement of blood CD4+ population and significant induction of specific immunoglobulin G1 (IgG1) and immunoglobulin G2a (IgG2a) antibodies indicated that SSVs and CSV2 induced not only humoral immunity but also T helper (Th) 1 T cell immunity. Finally, spleen proliferation and bacterial burden in the spleen of mice vaccinated with these subunit vaccines were significantly lower than those of PBS group, which conferred significant protection against B. abortus infection. The third chapter of this study investigated the combination of four recombinant B. abortus proteins, namely outer membrane protein 16 (Omp16), outer membrane protein (Omp19), outer membrane protein 28 (Omp28) and L7/L12, as combined subunit vaccine (CSV4) against B. abortus infection in vivo and in vitro. Immunoblotting assay showed that these four recombinant proteins, as well as pcold-TF vector reacted with Brucella¬-positive serum, but not with Brucella¬-negative serum. CSV4-treated RAW 264.7 cells significantly induced production of IFN-γ and interleukin 12 (IL-12) while decreased IL-10 production at the late stage of infection compared to PBS group. Enhancement of nitric oxide (NO) production together with cytokines secretion profile in CSV4-treated cells proved that CSV4 notably activated bactericidal mechanisms in macrophages. In consistent, mice immunized with CSV4 strongly elicited production of pro-inflammatory cytokine TNF-α, IL-6 and monocyte chemoattractant protein 1 (MCP-1) compared to PBS group. Moreover, concentration of IFN-γ was greater than IL-10 as well as higher titer of IgG2a compared to IgG1 in CSV4-immunized mice suggested that CSV4 induced predominantly Th1 T cell. Because of activation of strong immunity against intracellular Brucella, the superior protective effect conferred by this vaccine was observed with 1.41-log protection compared to PBS group. Collectively, this study provides protective effects of herbal medicine and subunit vaccine against Brucella infection. However, many studies are still necessary to screen new promising candidates, develop better subunit vaccines and herbal medicine in brucellosis prevention and treatment. 브루셀라증은 Brucella 균에 의해 발병되는 대표적인 인수공통전염병이다. 브루셀라균 감염은 전세계적으로 산업동물에서 경제적 손실과 공중보건학적 문제를 야기한다. 그러므로 다양한 방법의 제어법과 근절 전략이 인체 및 산업동물 브루셀라증에 적용되고 있다. 브루셀라증 치료를 위해 사용되고 있는 항생제의 사용이 제한되고 있는데, 이는 브루셀라균이 세포 내 기생균이며, 장기간의 치료에 따른 부작용, 항생제 내성 등이 문제가 되기 때문이다. 따라서, 백신을 이용한 질병의 예방과 천연소재를 이용한 항생제 대체 치료가 주목을 받고 있다. 본 연구의 첫 번째 내용은 다양한 천연소재에서 발견되고 있는 성분 중 하나인 Emodin이 브루셀라균의 대식구 감염에서 나타나는 치료효과를 규명하고자 수행하였다. 살균효력을 측정하기 위하여, 브루셀라균을 다양한 농도의 Emodin과 반응시켰다. 그 결과 농도가 높을수록, 반응시간을 길게 할수록 살균적이 증가하는 것을 확인 하였다. 브루셀라균의 세포 내 침입능력을 확인 하기 위하여, 세포독성을 보이지 않는 Emodin의 농도를 확립하였고, 세포독성을 보이지 않는 Emodin을 대식구에 처리 한 결과, 브루셀라균의 대식구 침입이 억제 되는 것을 확인 하였지만, 세포내 증식을 억제 하지 않는 것으로 나타났다. 또한, Emodin의 처리는 브루셀라균의 대식구내 부착을 억제 하는 것으로 나타났다. MAPK signaling과 F-actin polymerization을 측정하기 위하여 브루셀라균을 감염한 대식구에 대하여 immunoblotting과 FACS 분석을 수행해 본 결과 Emodin처리 대식구에서 ERK1/2의 인산화 정도가 감소 되었으며, F-actin polymerization 또한 비처리 세균에 비해 감소 되었다. 본 연구를 통해 Emodin이 동물 브루셀라증을 예방하기 위한 천연물질 대체제로 활용 가능성을 확인 하였다. 두 번째 내용은 브루셀라 감염증에 대한 예방 기법의 응용으로 브루셀라균의 Adk 항원과 SecB 재조합 항원을 이용한 subunit vaccine의 적용이다. 두 개의 재조합 단백을 작제 하였고, 각각의 유전자는 B. abortus균의 Adk와 SecB 유전자를 coding 하는 단백질이다. 이들 각각의 재조합 단백을 이용한 단일 백신과 혼합백신을 활용하여, 마우스에 예방효과를 수행하였다. 두개의 Adk와 SecB 유전자는 pcold-TF expression system에 clone 하였고, 각각의 재조합 단백질은 E. coli DH5α 대장균에 발현을 수행하였다. 정제된 재조합 단백질 중 재조합단백 Adk (rAdk)와 재조합단백 SecB (rSecB)은 각각 단일 백신과 rAdk와 rSecB를 혼합한 혼합 백신으로 평가하였다. 발현된 2개의 재조합 단백질의 면역원성을 규명하기 위하여, 각각의 재조합 단백은 정제 후 브루셀라 감염 혈청과 immunoblotting을 수행해본 결과 강한 면역반응을 보이는 것으로 확인 되었다. rAdk와 rSecB은 단일백신과 2종을 혼합한 혼합백신을 마우스에 접종면역하였으며, 음성 대조군으로 pcold-TF 단백과 PBS를 각각 면역하였고, 양성대조군으로 대표적인 vaccine strain인 RB51을 접종하여 백신효능을 평가하였다. 면역 후 혈중 형성 된 cytokines를 측정해본 결과, 단일 및 혼합 백신 모두에서 TNF-α와 IL-6같은 pro-inflammatory cytokine이 높게 발현되는 것이 확인 되어 innate immune response를 유도하는 것을 확인 하였다. 이는 초기 감염에 대식구의 항균 기전을 증가시키는 물질로 평가 할 수 있다. 한편, 단일 및 혼합 백신 모두에서 PBS 면역 음성 대조군과 비교하여 IFN-γ산생을 증가시키고, IL-10을 감소시키는 것으로 나타났다. 이는 IFN-γ과 IL-10이 IgG1과 IgG2a 발현에 영향을 주는 cytokine으로, 이들 두 cytokine의 발현 조절은 체액성 면역뿐만 아니라 Th1 T cell 면역에도 중요한 역할을 담당하는 것으로 사료된다. 또한 rAdk 및 rSecB 각각 면역에 의한 단일백신과 rAdk과 rSecB 혼합백신은 CD4+ T cell population을 유도하였다. 마우스 면역 후 수행한 공격접종을 통해 확인한 비장 내 균 수 및 비장증식은 PBS 면역 후 수행한 공격접종 군에 비하여 높은 수준의 감소를 확인 할 수 있어서, 재조합백신을 접종한 모든 그룹에서 우수한 브루셀라증 예방 효과를 확인 할 수 있었다. 세 번째 내용은 브루셀라 감염증에 대한 예방 기법의 응용으로 브루셀라균의 Omp16, Omp19, Omp28 및 L7/L12 재조합 항원을(rOmp16, rOmp19, rOmp28 및 rL7/L12) 이용한 subunit vaccine의 적용이다. 네 개의 재조합 단백을 작제 하였고, 각각의 유전자는 B. abortus 균의 Omp16, Omp19, Omp28 및 rplL 유전자를 coding 하는 단백질이다. 이들 각각의 재조합 단백을 이용한 혼합백신(CSV4)을 활용하여, 마우스에 예방효과를 수행하였다. 네개의 Omp16, Omp19, Omp28 및 rplL 유전자는 상기 2번째 내용과 같은 방법으로 재조합 단백발현을 수행하였고, 각각의 재조합 단백질의 면역원성을 규명하기 위하여, 정제 후 브루셀라 감염 혈청과 immunoblotting을 수행해본 결과 강한 면역반응을 보이는 것으로 확인 되었다. CSV4는 마우스유래 대식구 (RAW 264.7 cells) 처리 시 IFN-γ과 IL-12의 발현을 유도하지만 IL-10의 경우 PBS 접종한 음성대조군에 비하여 감소하는 것으로 나타났으며, 처리군에서 대식구내 살균에 영향을 미치는 NO 산생이 증가하는 것으로 나타났다. 혼합백신인 CSV4를 마우스 면역 후 pro-inflammatory cytokine인 TNF-α, IL-6와 MCP-1의 산생을 PBS 음성대조군에 비하여 강하게 유도하였으며, IFN-γ산생이 IL-10보다 증가하였고, IgG2a의 산생이 IgG1보다 우수한 것으로 나타나, CSV4의 면역은 마우스의 Th1 T cell을 유도하는 것으로 사료된다. 이렇듯 세포 내 기생세균인 브루셀라균에 대한 강한 면역반응 때문에 CSV4의 면역은 공격접종 후 확인 한 예방 효과 측정에서도 PBS 음성 대조군에 비하여 1.41-log protection을 확인. 종합적으로 본 연구는 천연소재 물질과 subunit 백신을 이용하여 브루셀라 감염증의 예방효과를 규명하였다. 브루셀라 감염증에 있어서, 높은 면역원성을 보이는 항원을 활용한 subunit 백신의 개발은 아직도 많은 연구가 필요한 상황이며, 치료용 신소재에 개발과 응용연구가 다양하게 진행 되어야 할 것이다.

랫드에서 di-n-butylphthalate로 유도된 고환독성에 대한 흑삼의 보호효과

박찬주 창원대학교 2023 국내석사

Di-n-butylphthalate (DBP) is a phthalate-based material that is used as a plasticizer to soften polyvinyl chloride (PVC) and is classified as an endocrine disruptor with antiandrogen effects. Exposure to DBP induces oxidative stress due to excessive production of reactive oxygen species (ROS) in rat testes, resulting in testicular toxicity. Black ginseng has higher antioxidant activity than white ginseng and red ginseng through repeated heat treatment and processing. This study investigated whether the antioxidant activity of black ginseng could protect testicular toxicity induced by DBP in adolescent rats. As a result of organ weight measurement after administration of the test substance, a significant decrease in testicular weight was found in DBP group (DBP 500 mg/kg bw/day) and B.G + DBP group (B.G 2.5 ml/kg bw/day + DBP 500 mg/kg bw/day, B.G 5 ml/kg bw/day + DBP 500 mg/kg bw/day, B.G 10 ml/kg bw/day + DBP 500 mg/kg bw/day), excluding B.G group (B.G 10 ml/kg bw/day), when compared to control group. Compared to DBP group, there was a significant weight difference in B.G 10 + DBP 500 group. The epididymal weight showed a significant decrease in DBP group and B.G 2.5 + DBP 500 group, when compared to control group. As a result of histopathological analysis, significant histopathological changes such as irregular arrangement and atrophy of seminiferous tubules, Sertoli cell and Leydig cell damage were observed in DBP group and B.G 2.5 + DBP 500 group. However, in control group and the other B.G + DBP group, no change in the shape of the seminiferous tubules of rats was observed. As a result of testosterone measurement, a significant decrease in serum testosterone concentration was found in DBP group compared to control group, and there was no significant difference in serum testosterone concentration in B.G + DBP group compared to control group. As a result of western blot, the expression levels of Nrf2 and Nrf2 sub-proteins HO-1 and NQO1 increased in DBP group compared to control group. Compared to DBP group, the expression levels of Nrf2, HO-1 and NQO1 proteins were decreased in the B.G + DBP group. In conclusion, B.G has a protective effect against testicular toxicity induced by DBP.

Rotundarpene이 파킨슨병 유사증상 유발 신경독소 1-methyl-4-phenylpyridinium에 의한 신경세포 아포토시스에 미치는 억제효과

한상우 中央大學校 大學院 2016 국내석사

Ilex Rotunda Thunb의 추출물과 헤미테르핀 배당체는 항염증, 항산화 효과를 가지고 있다. 그럼에도 1-methyl-4-phenylpyridinium에 의해 유발된 신경세포 아포토시스에 미치는 rotundarpene의 효과는 조사되지 않았다. 신경증식인자로 분화된PC12 세포와 사람 신경모세포종 세포주 SH-SY5Y 세포를 이용하여 1-methyl-4-phenylpyridinium에 의해 유발된 신경세포 아포토시스에 미치는 rotundarpene 효과를 세포사 과정의 관점에서 분석하였다. 1-methyl-4-phenylpyridinium은 세포질 Bid, Bcl-2 양의 감소, 세포질 Bax, p53 양의 증가, 미토콘드리아 막 전위의 소실, cytochrome c의 유리, caspases의 활성화 (-8, -9, -3)를 유발하였다. 분화된PC12 세포와 SH-SY5Y 세포에서 rotundarpene은 1-methyl-4-phenylpyridinium에 의해 유발된 아포토시스 관련 단백질 양의변화, 활성산소의 생성, GSH의 고갈과 산화, 세포사를 억제하였다. 연구결과로부터 신경세포에서 rotundarpene은 caspase 활성화에 이르는 미토콘드리아 매개 세포사 경로뿐만 아니라 caspase-8-Bid 경로 활성화를 억제하여 1-methyl-4-phenylpyridium에 의한 아포토시스를 억제한다고 생각한다. Rotundarpene이 아포토시스에 미치는 억제효과는 활성산소생성, 글루타티온 고갈과 산화의 억제에 의해 이루어졌을 것으로 생각한다. Rotundarpene은 파킨슨병 유사증상 유발 신경독소에 의해 이루어지는 신경 세포사를 억제할 수 있을 것으로 예상한다. The extract and hemiterpene glycosides of Ilex Rotunda Thunb have demonstrated antioxidant and anti-inflammatory effects. Nevertheless, the effect of rotundarpene on the 1-methyl-4-phenylpyridinium-induced apoptosis in neuronal cells has not been investigated. Using differentiated PC12 cells and human neuroblastoma SH-SY5Y cells, we investigated the effect of rotundarpene on 1-methyl-4-phenylpyridinium-induced apoptosis in relation to cell death process. 1-methyl-4-phenylpyridinium causes apoptosis by eliciting a decrease in the cytosolic levels of Bid and Bcl-2 proteins, increase in the cytosolic levels of Bax and p53, loss of the mitochondrial transmembrane potential, release of cytochrome c and activation of caspases (-8, -9 and -3) in differentiated PC12 cells. Treatment with rotundarpene prevented 1-methyl-4-phenylpyridinium-induced changes in the levels of apoptosis-related proteins, formation of reactive oxygen species, depletion and oxidation of GSH, and cell death in PC12 cells and SH-SY5Y cells. Rotundarpene may reduce 1-methyl-4-phenylpyridinium-induced apoptosis in PC12 cells by suppressing the activation of the mitochondria-mediated pathway and the caspase-8- and Bid-pathways. The effect of rotundarpene appears to be associated with its inhibitory effect on the formation of reactive oxygen species, and depletion and oxidation of GSH.