[논문]INS-1 췌장 베타 세포에서 벼메뚜기(Oxya chinensis sinuosa Mistshenk) 추출물의 당독성 개선 효과

박재은; 한지숙

doi:10.5352/jls.2021.31.11.969

INS-1 췌장 베타 세포에서 벼메뚜기(Oxya chinensis sinuosa Mistshenk) 추출물의 당독성 개선 효과
Oxya chinensis sinuosa Mishchenko (Grasshopper) Extract Protects INS-1 Pancreatic β cells against Glucotoxicity-induced Apoptosis and Oxidative Stress 원문보기

생명과학회지 = Journal of life science, v.31 no.11, 2021년, pp.969 - 979

초록
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당뇨병은 만성대사성 질환으로 우리나라 국민의 사망요인 중 5위를 차지하고 있다. 제 2형 당뇨병에서 나타나는 인슐린 분비 감소는 베타세포의 자가사멸에 의한 베타세포질량의 급격한 감소로 인한 것으로 보고되고 있으며, 베타세포의 자가사멸을 촉진하는 요인으로 고혈당에 의한 당독성 및 활성산소종들의 증강에 의한 산화스트레스 등이다. 벼메뚜기 추출물은 고농도 포도당 처리된 INS-1 췌장 베타 세포에서 세포 생존율을 증가시키고, 지질과산화물, 세포 내 ROS 및 NO 수준을 감소시켰다. 세포사멸 관련 인자의 유전자 발현 결과 pro-세포자가사멸인자인 Bax, Cytochrome C, caspase-3 및 caspase-9의 단백질 발현을 유의적으로 감소시켰고, anti-세포자가사멸인자인 Bcl-2 발현을 증가시켰다. Annexin V/I propidium iodide 염색법을 통하여 벼메뚜기 추출물이 고농도 포도당으로 유도된 세포 사멸을 감소시키고, INS-1 췌장 베타세포에서의 인슐린 분비능을 증가시키는 것으로 사료된다. 그러므로 벼메뚜기 추출물이 고농도 포도당으로 손상된 INS-1 췌장 베타세포의 보호효과를 나타낸다. 본 연구의 결과는 벼메뚜기 추출물이 당뇨병 치료에 도움을 주는 항당뇨 기능성 식품 소재 및 개발에 기여할 수 있음을 시사한다.

Abstract ▼ AI-Helper

Type 2 diabetes is a serious chronic metabolic disease, and the goal of diabetes treatment is to keep blood glucose at a normal level and prevent complications from diabetes. Hyperglycemia is a key pathologic feature of type 2 diabetes that mainly results from insulin resistance and pancreatic β-cell dysfunction. Chronic exposure of β-cells to elevated glucose concentrations induces glucotoxicity. In this study, we examined whether an 80% ethanol extract of Oxya chinensis sinuosa Mishchenko (OEE) protected INS-1 pancreatic β-cells against glucotoxicity-induced apoptosis and oxidative stress. Pretreatment with a high concentration of glucose (high glucose = 30 mM) induced glucotoxicity and apoptosis of INS-1 pancreatic β cells. Treatment with OEE significantly increased cell viability. Treatment with 0.01-0.20 mg/ml OEE dose dependently decreased intracellular reactive oxygen species, lipid peroxidation, and nitric oxide levels and increased insulin secretion in high glucose-pretreated INS-1 β cells. OEE also significantly increased the activities of antioxidant enzymes in response to high-glucose-induced oxidative stress. Moreover, OEE treatment significantly reduced the expressions of pro-apoptotic proteins, including Bax, cytochrome C, caspase-3, and caspase-9, and increased anti-apoptotic Bcl-2 expression. Apoptotic cells were identified using Annexin-V/propidium iodide staining, which revealed that treatment with OEE significantly reduced high-glucose-induced apoptosis. These findings implicate OEE as a valuable functional food in protecting pancreatic β-cells against glucotoxicity-induced apoptosis and oxidative stress.

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표/그림 (6)

그림 Fig. 1. Effect of OEE on cell viability in INS-1 β cells treated with a high concentration of glucose. INS-1 β cells (2×10⁴ per well) were pre-incubated in 96-well plates with 5.5 or 30 mM glucose for 48 hr. The cells were then incubated with 0.01, 0.05, 0.1, 0.15, and 0.2 mg/ml OEE for 48 hr. The 5.5 mM and 30 mM concentrations of glucose represents normal and high glucose levels, respectively . Each value is expressed as the mean ± SD (n=3). ^a-fValues are significantly different at p<0.05, as analyzed by Duncan's multiple-range test.
그림 Fig. 2. Effects of OEE on intracellular levels of ROS, TBARS, and NO in INS-1 β cells treated with a high concentration of glucose. INS-1 β cells (2×10⁴ per well) were pre-incubated in 96-well plates with 5.5 or 30 mM glucose for 48 hr, and then incubated with 0.01, 0.05, 0.1, 0.15, and 0.2 mg/ml OEE for 48 hr. The 5.5 mM and 30 mM concentrations of glucose represent normal and high glucose levels, respectively. (A) The effect of OEE on intracellular levels of ROS. (B) the effect of OEE on intracellular levels of TBARS. (C) The effect of OEE on intracellular levels of NO in high glucose-treated INS-1 cells. Each value is expressed as the mean ± SD (n=3). ^a-fValues are significantly different at p<0.05, as analyzed by Duncan’s multiple-range test.
표 Table 1. Effects of OEE antioxidant enzymes in high glucose-treated INS-1 cells
그림 Fig. 3. Effects of OEE on Bax, Bcl-2, cytochrome C, caspase-9, and caspase-3 protein expression in high glucose-treated INS-1 β cells. INS-1 β cells were pre-incubated in 96-well plates with 5.5 or 30 mM glucose for 48 hr, followed by 0.1 and 0.2 mg/ml OEE for 48 hr. The 5.5 mM and 30 mM concentrations of glucose represents normal and high glucose levels, respectively. Equal amounts of cell lysates were electrophoresed and analyzed for Bax, Bcl-2, cytochrome C, caspase 9, and caspase 3 protein using Western blots. β-actin was used as an internal control. (A) Bax, Bcl-2, cytochrome C, caspase-9, and caspase-3 protein expression. (B) expression levels of Bax, Bcl-2, cytochrome C, caspase-9, and caspase-3. Each value is expressed as the mean ± SD (n=3). ^a-dValues are significantly different at p<0.05, as analyzed by Duncan's multiple-range test.
그림 Fig. 4. Identification of the apoptotic stage of INS-1 β cells with different treatments using Annexin-V/PI staining. (A) Apoptotic cell death was determined by flow cytometry enumeration of INS-1 β cells stained with Annexin-V-fluorescein isothiocyanate/PI. Cells were pre-incubated with glucose and then incubated in the presence or absence of 0.1 and 0.2 mg/ml OEE. The right lower and upper quadrants show the numbers of early and late apoptotic cells, respectively. (B) Mean rates of apoptosis. ^a-dValues are significantly different at p<0.05, as analyzed Duncan's multiple-range test.
그림 Fig. 5. Effect of OEE on insulin secretion in high glucose-treated INS-1 β cells. INS-1 β cells (2×10⁴ per well) were pre-incubated in 96-well plates with 5.5 or 30 mM glucose for 48 hr, and then incubated with 0.01, 0.05, 0.1, 0.15, and 0.2 mg/ml OEE for 48 hr. Thereafter, the cells were stimulated with Krebs–Ringer buffer containing 5 mM glucose for 60 min. The 5.5 mM and 30 mM concentrations of glucose represent normal and high glucose levels, respectively. Each value is expressed as the mean ± SD (n=3). ^a-eValues are significantly different at p<0.05, as analyzed by Duncan's multiple-range test.

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데이터처리

Treatment groups were compared by one-way analysis of variance (ANOVA) followed by a post hoc Duncan’s multiple-range test

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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