WO2019088636A2 - Method for analyzing antioxidant effect of low dose radiation via fruit fly and method for enhancing antioxidation - Google Patents

Method for analyzing antioxidant effect of low dose radiation via fruit fly and method for enhancing antioxidation Download PDF

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WO2019088636A2
WO2019088636A2 PCT/KR2018/012969 KR2018012969W WO2019088636A2 WO 2019088636 A2 WO2019088636 A2 WO 2019088636A2 KR 2018012969 W KR2018012969 W KR 2018012969W WO 2019088636 A2 WO2019088636 A2 WO 2019088636A2
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oxidative stress
low dose
dose radiation
fruit fly
radiation
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WO2019088636A3 (en
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남선영
정해민
황수진
백윤미
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한국수력원자력 주식회사
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  • the present invention relates to a method for analyzing the antioxidative effect of low dose radiation through a fruit fly and to a method for enhancing the antioxidative effect of the low dose radiation by irradiating low dose radiation to the fruit fly, will be.
  • Drosophila has a short life span of about 2 to 3 months and is therefore of high value as a model of aging research, it is mainly used in a variety of biological studies (Partridge et al., (2011) Exp. Gerontol.).
  • Oxidative stress one of the major causes of aging, has been identified as a representative environmental factor not only for aging but also for aging diseases such as Alzheimer's disease (Moskalev, (2007) Biogerontology, Finkle & Hobrook, Is a state in which the active oxygen in the body is rapidly increased in the energy production process of the body and the oxidation balance in the human body is collapsed. This is one of the problems that are manifested due to the eating habit and the living environment of the modern people. As a result, the importance of protein homeostasis has been raised in order to prevent oxidative damage of proteins and cell organelles due to oxidative stress.
  • the oxidative stress environment can be induced by the medium supplemented with hydrogen peroxide (H 2 O 2 ) or paraquat.
  • H 2 O 2 hydrogen peroxide
  • Experimental methods to confirm the antioxidative action of Drosophila in the above environment have been reported previously (Sohal, (1996) Science, Biteau et al., (2011) Exp. Gerontol.).
  • An object of the present invention is to prevent aging by improving the antioxidative effect by irradiating eggs of fruit flies with low dose radiation.
  • Another object of the present invention is to provide a method for analyzing the antioxidative effect of low dose radiation.
  • the present invention provides a method for producing a fruit fly comprising: (a) irradiating eggs of fruit flies with low dose radiation; (b) culturing the fruit flies irradiated with the low dose radiation into an adult; (c) exposing the adult of the fruit fly to an oxidative stress environment; And (d) evaluating the oxidative stress of the adult exposed to the oxidative stress environment.
  • the step (a) is characterized by irradiating low dose radiation of 0.01 to 0.2 Gy.
  • the oxidative stress environment in step (c) is a medium containing less than 1% hydrogen peroxide.
  • the oxidative stress evaluation in the step (d) is performed by measuring at least one of the survival rate, motility, accumulation of reactive oxygen species in the body and activation of AKT / p70S6K protein do.
  • the present invention provides a method for enhancing antioxidative activity of fruit flies by irradiating low-dose radiation with eggs of fruit flies.
  • the low-dose radiation may be characterized by being 0.01 to 0.2 Gy.
  • the relationship between low dose radiation and oxidative stress can be grasped by analyzing the antioxidative effect of low dose radiation, and therapies for treating various diseases due to oxidative stress can be devised.
  • an effect of providing basic research data such as an oxidative stress evaluation method for verifying the oxidative stress defense effect.
  • Drosophila has an antioxidative effect even in an oxidative stress environment when irradiated with low doses of radiation.
  • Figure 1 shows the Drosophila survival rate in an oxidative stress environment.
  • Figure 2 shows Drosophila motility in an oxidative stress environment.
  • Figure 3 shows the amount of ROS accumulation in the Drosophila in an oxidative stress environment.
  • Figure 4 shows the activation of AKT and p70S6K in an oxidative stress environment.
  • a method of producing a fruit fly comprising: (a) irradiating eggs of fruit flies with low dose radiation; (b) culturing the fruit flies irradiated with the low dose radiation into an adult; (c) exposing the adult of the fruit fly to an oxidative stress environment; And (d) evaluating the oxidative stress of the adult exposed to the oxidative stress environment.
  • step (a) low dose radiation is irradiated to the embryo of wild type fruit flies (Oregon R). Preferably 0.01 to 0.2 Gy, and more preferably irradiated with a low dose radiation of 0.05 Gy.
  • step (b) eggs of Drosophila, irradiated with each radiation in the step (a), were incubated in an incubator at 25 ⁇ until they become adult.
  • step (c) male adult flies of the fruit flies are exposed to an oxidative stress environment.
  • the oxidative stress environment refers to an environment in a medium to which hydrogen peroxide (H 2 O 2 ) or paraquat is added.
  • the concentration in the medium may be 20 mmol / L and in the case of hydrogen peroxide (H 2 O 2 ) it may be less than 1%.
  • Drosophila is added to the medium of the oxidative stress environment in a ratio of 10 to 20 / vial.
  • AKT / p70S6K (AKT or p70S6K) protein Preferably for about 9 days in the measurement of survival rate, 72 hours in the measurement of motility and activation of AKT / p70S6K (AKT or p70S6K) protein, 0 to 72 hours in the measurement of reactive oxygen species accumulation in the body, Respectively.
  • Oxidative stress of the adult is evaluated in the step (d). Oxidative stress assessment can be assessed by measuring at least one of the survival rate, motility, accumulation of ROS in the body, and activation of AKT / p70S6K protein.
  • Figure 1 shows the Drosophila survival rate in an oxidative stress environment.
  • the survival rate of Drosophila was higher than that when low dose radiation was not irradiated. This means that Drosophila is resistant to oxidative stress by low dose radiation.
  • the survival rate of 10 Gy high dose radiation was lower than that of no radiation dose. This can be deduced from the fact that irradiation with high dose radiation is more vulnerable to oxidative stress than that without irradiation.
  • Figure 2 shows the mobility of fruit flies in an oxidative stress environment.
  • Drosophila survival experiment male adult flies cultured after low dose irradiation were exposed to an oxidative stress environment for 72 hours. Climbing ability test method was used to measure the mobility of each flies.
  • the climbing ability of Drosophila is about 68% when irradiated with low dose of 0.05 Gy, which is better than the climbing ability of about 48% without irradiation. This is similar to the climbing ability of about 78% in an environment without oxidative stress.
  • 10 Gy high dose radiation is irradiated, it can be confirmed that the mobility is lower than that when no radiation is irradiated.
  • FIG. 3 shows the accumulation of ROS-Reactive Oxygen Species in an oxidative stress environment.
  • ROS is one of the main causes of aging.
  • the accumulation of Drosophila in ROS was measured by DCF-DA microplate assay. Comparison of ROS accumulation by time of exposure to the oxidative stress environment indicates that the accumulation of ROS in the body due to oxidative stress is delayed when irradiated with 0.05 Gy low dose radiation.
  • the activation and phosphorylation degree of cell survival signal protein AKT (protein kinase B) and p70S6K were compared and analyzed. AKT and p70S6K proteins are decreased by oxidative stress. The results of the analysis can be seen in FIG. In the oxidative stress environment, the signal transmission pathway is activated by p-AKT and p-p70S6K when 0.05 Gy low-dose radiation is irradiated.
  • the above oxidative stress evaluation confirmed the effect of enhancing the resistance of Drosophila to oxidative stress, that is, the antioxidant-promoting effect upon irradiation at a low dose.
  • AKT signaling mechanism is related to oxidative stress defense mechanism by low dose radiation.
  • Low dose radiation 0.05 Gy and high dose radiation 10 Gy were irradiated to the embryo of wild type Drosophila (Oregon R) of less than 6 hours respectively.
  • the egg of the fruit fly irradiated with the above radiation is cultured in an incubator at 25 ⁇ until it becomes an adult.
  • a medium containing 1% hydrogen peroxide (H 2 O 2 ) induces an oxidative stress-like environment.
  • Adult male Drosophila is transferred to a medium containing 1% hydrogen peroxide (H 2 O 2 ) and exposed for 72 hours (20 vials).
  • Drosophila survival rate, motility, accumulation of reactive oxygen species in the body and activation of AKT / p70S6K protein in an oxidative stress environment are measured.

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Abstract

The present invention relates to a method for analyzing an antioxidant effect of low dose radiation via a fruit fly and a method for enhancing antioxidation. More specifically, the present invention relates to a method for analyzing an antioxidant effect of low dose radiation comprising the steps of: (a) irradiating eggs of a fruit fly with low dose radiation; (b) culturing the eggs of the fruit fly irradiated with the low dose radiation into adults; (c) exposing the adult fruit flies to an oxidative stress environment; and (d) evaluating the oxidative stress of the adults exposed to the oxidative stress environment, and a method for enhancing antioxidation of a fruit fly by irradiating eggs of the fruit fly with low dose radiation to enhance antioxidant activity. The present invention as above has effects in that the relationship between the low dose radiation and the oxidative stress can be understood, and basic research data can be provided by the method for analyzing an antioxidant effect of low dose radiation, and it can be confirmed that irradiating the fruit fly with the low dose irradiation has an antioxidant effect even in an oxidative stress environment.

Description

초파리를 통한 저선량 방사선의 항산화 효과 분석 방법 및 항산화 증진 방법Analysis of antioxidative effect of low doses of radiation on fruit flies and promotion of antioxidants
본 발명은 초파리를 통한 저선량 방사선의 항산화 효과 분석 방법 및 항산화 증진 방법에 관한 것으로서, 더욱 상세하게는 저선량 방사선을 조사하여 초파리의 항산화 능력을 증진시키고 이에 따른 저선량 방사선의 항산화 효과를 분석하는 방법에 관한 것이다.The present invention relates to a method for analyzing the antioxidative effect of low dose radiation through a fruit fly and to a method for enhancing the antioxidative effect of the low dose radiation by irradiating low dose radiation to the fruit fly, will be.
최근 100 mSv이하의 저선량방사선 조사에 의해 개체성장을 촉진시킬 수 있는 가능성 뿐만 아니라 초파리의 수명 연장 효과를 확인한 연구 결과가 다수 발표되고 있다(Zhikrevetskaya et al., (2015) PLoS One, Seong et al., (2011) Biogerontology, Moskalev, (2007) Biogerontology). 초파리는 약 2~3개월의 짧은 수명을 가지기 때문에 노화 연구의 모델로서 가치가 높기 때문에 다양한 생물학 연구에 주로 활용된다(Partridge et al., (2011) Exp. Gerontol.).Recently, a number of studies have been published confirming the effect of prolonging the lifespan of Drosophila as well as the possibility of promoting the growth of individuals by low-dose irradiation of 100 mSv or less (Zhikrevetskaya et al., (2015) PLoS One, Seong et al. , (2011) Biogerontology, Moskalev, (2007) Biogerontology). Because Drosophila has a short life span of about 2 to 3 months and is therefore of high value as a model of aging research, it is mainly used in a variety of biological studies (Partridge et al., (2011) Exp. Gerontol.).
노화의 주된 원인 중 하나인 산화 스트레스는, 노화뿐만 아니라 알츠하이머병과 같은 노인성 질환의 대표적인 환경적 요인으로 지목되고 있다.(Moskalev, (2007) Biogerontology, Finkle & Hobrook, (2000) Nature) 산화 스트레스란 인체의 에너지 생성과정에서 체내 활성산소가 급격히 증가하여 인체 내 산화 균형이 무너진 상태를 의미하며 이는 현대인들의 식습관과 생활환경으로 인해 다수 발현되고 있는 문제점 중 하나이다. 그 결과 산화 스트레스로 인해 단백질 및 세포 소기관의 산화적 손상을 방지하기 위해 단백질 항상성 유지의 중요성이 대두되고 있다. Oxidative stress, one of the major causes of aging, has been identified as a representative environmental factor not only for aging but also for aging diseases such as Alzheimer's disease (Moskalev, (2007) Biogerontology, Finkle & Hobrook, Is a state in which the active oxygen in the body is rapidly increased in the energy production process of the body and the oxidation balance in the human body is collapsed. This is one of the problems that are manifested due to the eating habit and the living environment of the modern people. As a result, the importance of protein homeostasis has been raised in order to prevent oxidative damage of proteins and cell organelles due to oxidative stress.
초파리를 이용한 실험에 있어서 과산화수소(Hydrogen peroxide, H2O2) 혹은 파라쿼트(Paraquat) 등을 첨가한 배지로 산화 스트레스 환경을 유도할 수 있다. 상기한 환경에서 초파리의 항산화 반응을 확인할 수 있는 실험 방법은 앞서 보고된 바 있다(Sohal, (1996) Science, Biteau et al., (2011) Exp. Gerontol.).In the experiment using Drosophila, the oxidative stress environment can be induced by the medium supplemented with hydrogen peroxide (H 2 O 2 ) or paraquat. Experimental methods to confirm the antioxidative action of Drosophila in the above environment have been reported previously (Sohal, (1996) Science, Biteau et al., (2011) Exp. Gerontol.).
다만, 상기한 바와 같이 저선량 방사선에 의해 초파리의 수명이 증가하였다는 보고는 다수 있으나, 노화의 주된 원인으로 여겨지는 산화 스트레스와 저선량방사선의 연관성에 대한 연구는 아직 이루어지지 않아 초파리 실험 모델에서 저선량 방사선 조사와 산화 스트레스 유도를 동시에 진행한 연구 사례가 부족하다. 그 결과 방사선 조사 시기 및 적정 선량, 산화 스트레스 유도시간 등 기초 자료가 부족하여 유사한 연구과제 해결에 있어서도 어려움이 있다.However, there are many reports that the lifespan of Drosophila is increased by low-dose radiation. However, studies on the relationship between oxidative stress and low-dose radiation, which are considered to be the main causes of senescence, There is a lack of research examples that simultaneously investigate and induce oxidative stress. As a result, it is difficult to solve similar research problems due to lack of basic data such as irradiation time, proper dose, and induction time of oxidative stress.
[선행기술문헌][Prior Art Literature]
[비특허문헌][Non-Patent Document]
1. Zhikrevetskaya et al., (2015) PLoS One1. Zhikrevetskaya et al., (2015) PLoS One
2. Seong et al., (2011) Biogerontology2. Seong et al., (2011) Biogerontology
3. Moskalev, (2007) Biogerontology3. Moskalev, (2007) Biogerontology
4. Finkle & Hobrook, (2000) Nature4. Finkle & Hobrook, (2000) Nature
5. Sohal, (1996) Science5. Sohal, (1996) Science
6. Biteau et al., (2011) Exp. Gerontol.6. Biteau et al., (2011) Exp. Gerontol.
7. Partridge et al., (2011) Exp. Gerontol.7. Partridge et al., (2011) Exp. Gerontol.
8. Hwang et al., (2013) PLoS Genet.8. Hwang et al., (2013) PLoS Genet.
9. Chaudhuri et al., (2007) J. Neurosci.9. Chaudhuri et al., (2007) J. Neurosci.
10. Ma et al., (2016) Int. J. Mol. Med. 10. Ma et al., (2016) Int. J. Mol. Med.
11. Saberi et al., (2008) Am. J. Physiol. Cell Physiol. 11. Saberi et al., (2008) Am. J. Physiol. Cell Physiol.
본 발명의 목적은, 초파리의 알에 저선량 방사선을 조사함으로써, 항산화 효과를 증진시켜 노화를 방지함에 있다.An object of the present invention is to prevent aging by improving the antioxidative effect by irradiating eggs of fruit flies with low dose radiation.
본 발명의 다른 목적은 저선량 방사선의 항산화 효과 분석 방법을 제공함에 있다.Another object of the present invention is to provide a method for analyzing the antioxidative effect of low dose radiation.
상기 목적을 달성하기 위하여, 본 발명은 (a) 초파리의 알에 저선량 방사선을 조사하는 단계; (b) 상기 저선량 방사선을 조사한 초파리의 알을 성체로 배양시키는 단계; (c) 상기 초파리의 성체를 산화 스트레스 환경에 노출시키는 단계; 및 (d) 상기 산화 스트레스 환경에 노출된 성체의 산화 스트레스를 평가하는 단계;를 포함하는 저선량 방사선의 항산화 효과 분석방법을 제공한다.In order to accomplish the above object, the present invention provides a method for producing a fruit fly comprising: (a) irradiating eggs of fruit flies with low dose radiation; (b) culturing the fruit flies irradiated with the low dose radiation into an adult; (c) exposing the adult of the fruit fly to an oxidative stress environment; And (d) evaluating the oxidative stress of the adult exposed to the oxidative stress environment.
본 발명의 일실시예에 따르면 상기 (a) 단계는 0.01 내지 0.2 Gy의 저선량 방사선을 조사하는 것을 특징으로 한다.According to an embodiment of the present invention, the step (a) is characterized by irradiating low dose radiation of 0.01 to 0.2 Gy.
본 발명의 다른 일실시예에 따르면 상기 (c) 단계의 산화 스트레스 환경은 1%이하의 과산화수소를 포함한 배지인 것을 특징으로 한다.According to another embodiment of the present invention, the oxidative stress environment in step (c) is a medium containing less than 1% hydrogen peroxide.
본 발명의 또 다른 일실시예에 따르면 상기 (d) 단계의 산화 스트레스 평가는 초파리의 생존율, 운동성, 체내 활성산소종 축적량 및 AKT/p70S6K 단백질의 활성화 중 적어도 하나 이상을 측정하여 평가하는 것을 특징으로 한다.According to another embodiment of the present invention, the oxidative stress evaluation in the step (d) is performed by measuring at least one of the survival rate, motility, accumulation of reactive oxygen species in the body and activation of AKT / p70S6K protein do.
또한, 상기 목적을 달성하기 위하여, 본 발명은 저선량 방사선을 초파리의 알에 조사하여 항산화 활성을 증진시키는 초파리 항산화 증진 방법을 제공한다.In order to achieve the above object, the present invention provides a method for enhancing antioxidative activity of fruit flies by irradiating low-dose radiation with eggs of fruit flies.
본 발명의 일실시예에 따르면 상기 저선량 방사선은 0.01 내지 0.2 Gy 인 것을 특징으로 할 수 있다.According to an embodiment of the present invention, the low-dose radiation may be characterized by being 0.01 to 0.2 Gy.
상기와 같은 본 발명에 따르면, 저선량 방사선의 항산화 효과 분석방법에 의해 저선량 방사선과 산화 스트레스 간의 관계를 파악할 수 있고 그를 이용하여 산화 스트레스로 인한 다양한 질환의 치료법을 고안해낼 수 있다. 또한, 산화 스트레스 방어 효과 검증을 위한 산화 스트레스 평가 방법 등 기초 연구 자료를 제공할 수 있는 효과가 있다.According to the present invention as described above, the relationship between low dose radiation and oxidative stress can be grasped by analyzing the antioxidative effect of low dose radiation, and therapies for treating various diseases due to oxidative stress can be devised. In addition, there is an effect of providing basic research data such as an oxidative stress evaluation method for verifying the oxidative stress defense effect.
이와 더불어 초파리에게 저선량 방사선 조사시 산화 스트레스 환경에서도 항산화 효과가 있다.In addition, Drosophila has an antioxidative effect even in an oxidative stress environment when irradiated with low doses of radiation.
도 1은 산화 스트레스 환경에서 초파리 생존율을 도시한 것이다.Figure 1 shows the Drosophila survival rate in an oxidative stress environment.
도 2는 산화 스트레스 환경에서 초파리 운동성을 도시한 것이다.Figure 2 shows Drosophila motility in an oxidative stress environment.
도 3은 산화 스트레스 환경에서 초파리 체내 ROS 축적량을 도시한 것이다.Figure 3 shows the amount of ROS accumulation in the Drosophila in an oxidative stress environment.
도 4는 산화 스트레스 환경에서 AKT 및 p70S6K의 활성화를 도시한 것이다.Figure 4 shows the activation of AKT and p70S6K in an oxidative stress environment.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 형태에 따르면 (a) 초파리의 알에 저선량 방사선을 조사하는 단계; (b) 상기 저선량 방사선을 조사한 초파리의 알을 성체로 배양시키는 단계; (c) 상기 초파리의 성체를 산화 스트레스 환경에 노출시키는 단계; 및 (d) 상기 산화 스트레스 환경에 노출된 성체의 산화 스트레스를 평가하는 단계;를 포함하는 저선량 방사선의 항산화 효과 분석방법을 제공한다.According to one aspect of the present invention, there is provided a method of producing a fruit fly comprising: (a) irradiating eggs of fruit flies with low dose radiation; (b) culturing the fruit flies irradiated with the low dose radiation into an adult; (c) exposing the adult of the fruit fly to an oxidative stress environment; And (d) evaluating the oxidative stress of the adult exposed to the oxidative stress environment.
상기 (a) 단계에서 야생형 초파리(Oregon R)의 알(embryo)에 저선량 방사선을 조사한다. 바람직하게는 0.01 내지 0.2Gy의 저선량 방사선을 조사하고 더욱 바람직하게는 0.05 Gy의 저선량 방사선을 조사한다.In step (a), low dose radiation is irradiated to the embryo of wild type fruit flies (Oregon R). Preferably 0.01 to 0.2 Gy, and more preferably irradiated with a low dose radiation of 0.05 Gy.
상기 (b) 단계에서는 상기 (a) 단계에서 방사선을 각각 조사한 초파리의 알을 25℃ 인큐베이터에서 성체가 될 때까지 배양하였다.In the step (b), eggs of Drosophila, irradiated with each radiation in the step (a), were incubated in an incubator at 25 캜 until they become adult.
상기 (c) 단계에서는 상기 초파리의 성체 중 수컷 초파리를 산화 스트레스 환경에 노출시킨다. 상기 산화 스트레스 환경이란 과산화수소(Hydrogen peroxide, H2O2) 혹은 파라쿼트(Paraquat)를 첨가한 배지 내 환경을 의미한다. 파라쿼트의 경우 배지 내 농도가 20mmol/L일 수 있고, 과산화수소(Hydrogen peroxide, H2O2)의 경우 1% 이하일 수 있다. 산화 스트레스 환경의 배지에 초파리 성체를 10 내지 20 마리/vial의 비율로 첨가한다. 바람직하게는 생존율 측정시 약 9일 동안, 운동성 및 AKT/p70S6K(AKT 혹은 p70S6K) 단백질의 활성화 측정시 72시간 동안, 체내 활성산소종 축적량 측정시 0 내지 72시간 동안 초파리 성체를 산화 스트레스 환경에 노출시키며 측정하였다.In step (c), male adult flies of the fruit flies are exposed to an oxidative stress environment. The oxidative stress environment refers to an environment in a medium to which hydrogen peroxide (H 2 O 2 ) or paraquat is added. In the case of the paraquat, the concentration in the medium may be 20 mmol / L and in the case of hydrogen peroxide (H 2 O 2 ) it may be less than 1%. Drosophila is added to the medium of the oxidative stress environment in a ratio of 10 to 20 / vial. Preferably for about 9 days in the measurement of survival rate, 72 hours in the measurement of motility and activation of AKT / p70S6K (AKT or p70S6K) protein, 0 to 72 hours in the measurement of reactive oxygen species accumulation in the body, Respectively.
상기 (d) 단계에서 성체의 산화 스트레스를 평가한다. 산화 스트레스 평가는 초파리의 생존율, 운동성, 체내 활성산소종(ROS) 축적량 및 AKT/p70S6K 단백질의 활성화 중 적어도 하나 이상을 측정하여 평가할 수 있다.The oxidative stress of the adult is evaluated in the step (d). Oxidative stress assessment can be assessed by measuring at least one of the survival rate, motility, accumulation of ROS in the body, and activation of AKT / p70S6K protein.
도 1에서 산화 스트레스 환경에서의 초파리 생존율을 도시한다. 0.05 Gy의 저선량 방사선을 조사한 경우 저선량 방사선을 조사하지 않을 때에 비해 초파리의 생존율이 높은 것을 확인할 수 있다. 이는 저선량 방사선에 의해 초파리가 산화 스트레스에 대한 저항성을 가지게 됨을 의미한다. 반면 10 Gy의 고선량 방사선을 조사한 경우 방사선을 조사하지 않을 때보다 생존율이 낮아졌다. 이는 고선량 방사선을 조사하면 조사하지 않을 때보다 초파리가 산화 스트레스에 더 취약해짐을 유추할 수 있다.Figure 1 shows the Drosophila survival rate in an oxidative stress environment. When 0.05 Gy low-dose radiation was irradiated, the survival rate of Drosophila was higher than that when low dose radiation was not irradiated. This means that Drosophila is resistant to oxidative stress by low dose radiation. On the other hand, the survival rate of 10 Gy high dose radiation was lower than that of no radiation dose. This can be deduced from the fact that irradiation with high dose radiation is more vulnerable to oxidative stress than that without irradiation.
도 2는 산화 스트레스 환경 내의 초파리의 운동성을 도시한 것이다. 상기 초파리 생존율 실험과 동일하게 저선량 방사선 조사 후 배양한 성체 초파리 중 수컷을 72시간 동안 산화 스트레스 환경에 노출시킨다. Climbing ability test 방법을 이용하여 각 초파리의 운동성을 측정하였다. 산화 스트레스 환경 내에서 0.05 Gy의 저선량 방사선을 조사한 경우 초파리의 climbing ability는 대략 68%로 저선량 방사선을 조사하지 않은 경우의 climbing ability인 약 48%에 비해 개선됨을 확인할 수 있다. 이는 산화 스트레스가 없는 환경에서의 climbing ability인 약 78%와 유사하다. 반면 10 Gy의 고선량 방사선을 조사한 경우 방사선을 조사하지 않을 때보다 운동성이 낮아짐을 확인할 수 있다.Figure 2 shows the mobility of fruit flies in an oxidative stress environment. As in the Drosophila survival experiment, male adult flies cultured after low dose irradiation were exposed to an oxidative stress environment for 72 hours. Climbing ability test method was used to measure the mobility of each flies. In the oxidative stress environment, the climbing ability of Drosophila is about 68% when irradiated with low dose of 0.05 Gy, which is better than the climbing ability of about 48% without irradiation. This is similar to the climbing ability of about 78% in an environment without oxidative stress. On the other hand, when 10 Gy high dose radiation is irradiated, it can be confirmed that the mobility is lower than that when no radiation is irradiated.
도 3은 산화 스트레스 환경에서 초파리 체내 활성산소종(ROS-Reactive Oxygen Species)의 축적량을 도시한 것이다. ROS는 노화의 주된 원인으로 지목되고 있는 원인 중 하나이다. 이러한 ROS의 초파리 체내 축적량을 DCF-DA microplate assay를 통해 측정하였다. 산화 스트레스 환경에 노출된 시간대별로 ROS 축적량을 비교한 결과 0.05 Gy의 저선량 방사선을 조사한 경우, 산화 스트레스에 의한 체내 ROS 축적이 지연되는 것을 확인할 수 있다.Figure 3 shows the accumulation of ROS-Reactive Oxygen Species in an oxidative stress environment. ROS is one of the main causes of aging. The accumulation of Drosophila in ROS was measured by DCF-DA microplate assay. Comparison of ROS accumulation by time of exposure to the oxidative stress environment indicates that the accumulation of ROS in the body due to oxidative stress is delayed when irradiated with 0.05 Gy low dose radiation.
산화 스트레스 환경에서 저선량 방사선의 신호전달경로를 확인하기 위해 Cell survival 신호 단백질인 AKT(단백질인산화효소B)와 p70S6K의 활성화 즉 인산화 정도를 비교, 분석하였다. AKT와 p70S6K 단백질은 산화 스트레스에 의해 활성도가 감소되기 때문이다. 분석결과는 도 4에서 확인할 수 있다. 산화 스트레스 환경에서도 0.05Gy의 저선량 방사선을 조사한 경우 p-AKT, p-p70S6K로 인해 신호전달경로가 활성화됨을 알 수 있다.In order to confirm the pathway of low - dose radiation in oxidative stress environment, the activation and phosphorylation degree of cell survival signal protein AKT (protein kinase B) and p70S6K were compared and analyzed. AKT and p70S6K proteins are decreased by oxidative stress. The results of the analysis can be seen in FIG. In the oxidative stress environment, the signal transmission pathway is activated by p-AKT and p-p70S6K when 0.05 Gy low-dose radiation is irradiated.
상기 산화 스트레스 평가를 통해 저선량 방사선 조사시 산화 스트레스에 대한 초파리의 저항성 증진 효과 즉 항산화 증진 효과를 확인할 수 있었다. 또한, 저선량 방사선에 의한 산화 스트레스 방어 기작에 AKT 신호 기작이 연관되어 있음을 확인하였다.The above oxidative stress evaluation confirmed the effect of enhancing the resistance of Drosophila to oxidative stress, that is, the antioxidant-promoting effect upon irradiation at a low dose. In addition, it was confirmed that AKT signaling mechanism is related to oxidative stress defense mechanism by low dose radiation.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예 1. 저선량 방사선의 항산화 효과 분석Example 1. Antioxidative Effect Analysis of Low-dose Radiation
6시간 이하의 야생형 초파리(Oregon R)의 알(embryo)에 각각 저선량 방사선 0.05 Gy와 고선량 방사선 10 Gy(137Cs, 0.0159 Gy/s)를 조사하였다.Low dose radiation 0.05 Gy and high dose radiation 10 Gy ( 137 Cs, 0.0159 Gy / s) were irradiated to the embryo of wild type Drosophila (Oregon R) of less than 6 hours respectively.
상기 방사선을 조사한 초파리의 알을 25℃ 인큐베이터에서 성체가 될 때까지 배양한다.The egg of the fruit fly irradiated with the above radiation is cultured in an incubator at 25 캜 until it becomes an adult.
1% 과산화수소(Hydrogen peroxide, H2O2)를 포함한 배지로 산화 스트레스와 유사한 환경을 유도한다. 성체 수컷 초파리를 1% 과산화수소(Hydrogen peroxide, H2O2)를 포함한 배지로 옮겨 72시간동안 노출시킨다(20마리/vial).A medium containing 1% hydrogen peroxide (H 2 O 2 ) induces an oxidative stress-like environment. Adult male Drosophila is transferred to a medium containing 1% hydrogen peroxide (H 2 O 2 ) and exposed for 72 hours (20 vials).
산화 스트레스 환경에서의 초파리 생존율, 운동성, 체내 활성산소종 축적량, AKT/p70S6K 단백질의 활성화를 측정한다. Drosophila survival rate, motility, accumulation of reactive oxygen species in the body and activation of AKT / p70S6K protein in an oxidative stress environment are measured.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

  1. (a) 초파리의 알에 저선량 방사선을 조사하는 단계;(a) irradiating eggs of fruit flies with low dose radiation;
    (b) 상기 저선량 방사선을 조사한 초파리의 알을 성체로 배양시키는 단계;(b) culturing the fruit flies irradiated with the low dose radiation into an adult;
    (c) 상기 초파리의 성체를 산화 스트레스 환경에 노출시키는 단계; 및(c) exposing the adult of the fruit fly to an oxidative stress environment; And
    (d) 상기 산화 스트레스 환경에 노출된 성체의 산화 스트레스를 평가하는 단계;를 포함하는 저선량 방사선의 항산화 효과 분석방법.(d) evaluating the oxidative stress of the adult exposed to the oxidative stress environment.
  2. 제 1항에 있어서,The method according to claim 1,
    상기 (a) 단계는 0.01 내지 0.2 Gy의 저선량 방사선을 조사하는 것을 특징으로 하는 저선량 방사선의 항산화 효과 분석방법.Wherein the step (a) is a step of irradiating a low dose of 0.01 to 0.2 Gy of radiation.
  3. 제 1항에 있어서,The method according to claim 1,
    상기 (c) 단계의 산화 스트레스 환경은 과산화수소를 포함하되, 1% 이하의 과산화수소를 포함한 배지인 것을 특징으로 하는 저선량 방사선의 항산화 효과 분석방법.Wherein the oxidative stress environment in step (c) is a medium containing hydrogen peroxide and containing less than 1% hydrogen peroxide.
  4. 제 1항에 있어서,The method according to claim 1,
    상기 (d) 단계의 산화 스트레스 평가는 초파리의 생존율, 운동성, 체내 활성산소종 축적량 및 AKT/p70S6K 단백질의 활성화 중 적어도 하나 이상을 측정하여 평가하는 것을 특징으로 하는 저선량 방사선의 항산화 효과 분석방법.Wherein the oxidative stress evaluation in step (d) comprises measuring and evaluating at least one of survival rate, motility, accumulation of reactive oxygen species in the body, and activation of AKT / p70S6K protein.
  5. 저선량 방사선을 초파리의 알에 조사하여 항산화 활성을 증진시키는 초파리 항산화 증진 방법.Drosophila antioxidant enhancement method for enhancing antioxidant activity by irradiating low dose radiation to eggs of Drosophila.
  6. 제 5항에 있어서,6. The method of claim 5,
    상기 저선량 방사선은 0.01 내지 0.2 Gy 인 것을 특징으로 하는 초파리 항산화 증진 방법.Wherein said low dose radiation is between 0.01 and 0.2 Gy.
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