KR102623664B1 - Composition and treatment method for diabetic Alzheimer's disease using urolitin A - Google Patents
Composition and treatment method for diabetic Alzheimer's disease using urolitin A Download PDFInfo
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- KR102623664B1 KR102623664B1 KR1020200172223A KR20200172223A KR102623664B1 KR 102623664 B1 KR102623664 B1 KR 102623664B1 KR 1020200172223 A KR1020200172223 A KR 1020200172223A KR 20200172223 A KR20200172223 A KR 20200172223A KR 102623664 B1 KR102623664 B1 KR 102623664B1
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- urolithin
- cells
- high glucose
- tgm2
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Abstract
본 발명은 유로리틴 A를 이용한 당뇨성 알츠하이머병의 치료 조성물 및 치료 방법에 관한 것으로, 구체적으로 고혈당 또는 고포도당 조건으로 유도된 미토콘드리아 활성산소종의 발생을 감소시키거나, 미토콘드리아 칼슘 축적을 억제하고, 아밀로이드 베타의 생성 및 뉴런 변성을 억제하는 조성물에 관한 것이다.The present invention relates to a composition and treatment method for diabetic Alzheimer's disease using urolithin A, and specifically, to reduce the generation of mitochondrial reactive oxygen species induced by high blood sugar or high glucose conditions, or to inhibit mitochondrial calcium accumulation; It relates to a composition that inhibits the production of amyloid beta and neuronal degeneration.
Description
본 발명은 유로리틴 A를 이용한 당뇨성 알츠하이머병의 치료 조성물 및 치료 방법에 관한 것으로, 구체적으로 고혈당 또는 고포도당 조건으로 유도된 미토콘드리아 활성산소종의 발생을 감소시키거나, 미토콘드리아 칼슘 축적을 억제하고, 아밀로이드 베타의 생성 및 뉴런 변성을 억제하는 조성물에 관한 것이다.The present invention relates to a composition and treatment method for diabetic Alzheimer's disease using urolithin A, and specifically, to reduce the generation of mitochondrial reactive oxygen species induced by high blood sugar or high glucose conditions, or to inhibit mitochondrial calcium accumulation; It relates to a composition that inhibits the production of amyloid beta and neuronal degeneration.
당뇨병 (DM) 환자의 고혈당증은 신경 세포 손상, 신경 퇴행 및 인지 장애와 같은 아밀로이드 생성이 유도된 신경병리학적 변화에 기여하는 주요 병원성 요인이다. 이전 연구에서는 고농도의 포도당 조건이 신경 세포에서 아밀로이드 전구체 단백질 (APP) 축적과 아밀로이드 베타 (Aβ) 플라크 형성을 증가시키는 것으로 나타났다. 높은 포도당에 의한 APP 처리 효소 β-세크레타제 1 (BACE1)의 발현 증가는 신경 세포의 Aβ 생산에 중요하며, BACE1의 비활성화는 시냅스 및 기억력 결핍과 같은 Aβ 기반의 알츠하이머병(AD) 유사 병증을 완화한다. 인지 장애 등을 포함하는 고혈당으로 유발된 당뇨병 합병증은 주로 산화 스트레스와 관련되어 있다. 미토콘드리아 활성 산소종(mtROS)은 미토콘드리아 기능 장애, RNA 및 DNA 손상, 지질 과산화 및 Aβ 산화를 촉진하여 AD의 발병 기전에 핵심적인 역할을 한다. mtROS는 아밀로이드 생성 및 신경 세포 자멸사에 관여하기 때문에, 고혈당 상태에서 mtROS 축적을 억제하는 것은 DM 환자에서 AD 관련 질병을 예방할 수 있는 전략이라 볼 수 있다.Hyperglycemia in diabetes mellitus (DM) patients is a major pathogenic factor contributing to amyloidogenesis-induced neuropathological changes, such as neuronal damage, neurodegeneration, and cognitive impairment. Previous studies have shown that high glucose conditions increase amyloid precursor protein (APP) accumulation and amyloid beta (Aβ) plaque formation in neurons. Increased expression of the APP processing enzyme β-secretase 1 (BACE1) by high glucose is important for Aβ production in neurons, and inactivation of BACE1 causes Aβ-based Alzheimer's disease (AD)-like pathologies such as synaptic and memory deficits. alleviate. Hyperglycemia-induced diabetes complications, including cognitive impairment, are mainly related to oxidative stress. Mitochondrial reactive oxygen species (mtROS) play a key role in the pathogenesis of AD by promoting mitochondrial dysfunction, RNA and DNA damage, lipid peroxidation, and Aβ oxidation. Since mtROS is involved in amyloid production and neuronal apoptosis, inhibiting mtROS accumulation under hyperglycemic conditions can be considered a strategy to prevent AD-related diseases in DM patients.
고혈당증에 의한 미토콘드리아 칼슘 항상성의 조절 장애는 mtROS 축적의 위험 인자이며, 이는 당뇨병성 신경 병증을 유발한다. 미토콘드리아 관련 소포체 (ER) 막 (MAM)은 ER에서 미토콘드리아로의 세포 대사와 칼슘 수송을 제어하는 미토콘드리아와 ER 사이의 기관이다. Aβ-펩티드가 증가된 MAM 의존성 칼슘 수송은 AD에 관여하는 1차 해마 뉴런에서 전압 의존성 음이온 선택 채널 단백질 1 (VDAC1)-이노시톨 1, 4, 5- 트리스 포스페이트 수용체 (IP3R) 가교 형성에 의해 매개된다. GRP75, Bax, Bcl2, TGM2와 같은 IP3R-VDAC1 상호 작용을 조절하는 MAM의 조절 인자가 연구되고 있다.Hyperglycemia-induced dysregulation of mitochondrial calcium homeostasis is a risk factor for mtROS accumulation, which leads to diabetic neuropathy. The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is an organelle between mitochondria and the ER that controls cellular metabolism and calcium transport from the ER to the mitochondria. Aβ-peptide-enhanced MAM-dependent calcium transport is mediated by voltage-dependent anion selective channel protein 1 (VDAC1)-inositol 1,4,5-tris phosphate receptor (IP3R) cross-link formation in primary hippocampal neurons implicated in AD. . Regulators of MAM that regulate IP3R-VDAC1 interaction, such as GRP75, Bax, Bcl2, and TGM2, are being studied.
한편, 3,8-Dihydroxy-유로리틴 (유로리틴 A)은 장내 미생물에서 생성되는 엘라지타닌의 대사 산물이다. 엘라지타닌과 엘라그산에서 추출한 유로리틴의 생체 이용률은 개별 장내 미생물 전체 구성에 따라 상이하며, 유로리틴의 활성에 관한 종래 연구에서는 유로리틴 A가 전구체인 엘라지타닌과 다른 유로리틴에 비해 활성이 높은 대사 산물이라는 것을 연구하였다(J Ethnopharmacol. 2014;155:801-9.). 그러나 유로리틴 A가 고혈당 조건하에서 신경 세포에서 mtROS 생성을 조절하는 ER로부터 MAM 형성 및 미토콘드리아 칼슘 유입에 미치는 구체적인 영향에 대해서는 연구된 바가 없는 실정이다.Meanwhile, 3,8-Dihydroxy-urolithin (urolithin A) is a metabolite of ellagitannin produced in intestinal microorganisms. The bioavailability of urolithin extracted from ellagitannin and ellagic acid varies depending on the overall composition of individual intestinal microorganisms, and previous studies on the activity of urolithin showed that urolithin A was more active than its precursor, ellagitannin, and other urolithins. It was studied that it is a high metabolite (J Ethnopharmacol. 2014;155:801-9.). However, the specific effects of urolithin A on MAM formation and mitochondrial calcium influx from the ER, which regulates mtROS production in neurons under high glucose conditions, have not been studied.
본 발명의 하나의 목적은, 유로리틴 A를 유효성분으로 포함하는, 알츠하이머병의 예방 또는 치료용 약학 조성물을 제공하는 것이다.One object of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease, containing urolithin A as an active ingredient.
본 발명의 다른 하나의 목적은, 유로리틴 A를 유효성분으로 포함하는, 알츠하이머병의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a health functional food composition for preventing or improving Alzheimer's disease, containing urolithin A as an active ingredient.
본 발명의 다른 하나의 목적은, 후보물질 또는 유로리틴 A를 개체에 처리하는 단계; 개체의 미토콘드리아 활성산소종, LDH 생성, TGM2 발현량 또는 미토콘드리아 칼슘 축적의 정도를 비교하는 단계를 포함하는, 알츠하이머병의 예방 또는 치료 후보 물질을 스크리닝하는 방법을 제공하는 것이다.Another object of the present invention is to treat a subject with a candidate substance or urolithin A; The present invention provides a method for screening candidates for the prevention or treatment of Alzheimer's disease, which includes comparing the degree of mitochondrial reactive oxygen species, LDH production, TGM2 expression, or mitochondrial calcium accumulation in the subject.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는 유로리틴 A를 유효성분으로 포함하는, 알츠하이머병의 예방 또는 치료용 약학 조성물을 제공한다.One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising urolithin A as an active ingredient.
본 발명의 다른 하나의 양태는 유로리틴 A를 유효성분으로 포함하는, 알츠하이머병의 예방 또는 개선용 건강기능식품 조성물을 제공한다.Another aspect of the present invention provides a health functional food composition for preventing or improving Alzheimer's disease, comprising urolithin A as an active ingredient.
본 발명의 다른 하나의 양태는 후보물질 또는 유로리틴 A를 개체에 처리하는 단계; 개체의 미토콘드리아 활성산소종, LDH 생성, TGM2 발현량 또는 미토콘드리아 칼슘 축적의 정도를 비교하는 단계를 포함하는, 알츠하이머병의 예방 또는 치료 후보 물질을 스크리닝하는 방법을 제공한다.Another aspect of the present invention includes treating a subject with a candidate substance or urolithin A; Provided is a method for screening candidates for the prevention or treatment of Alzheimer's disease, comprising comparing the degree of mitochondrial reactive oxygen species, LDH production, TGM2 expression, or mitochondrial calcium accumulation of the subject.
본 발명에서는 유로리틴A가 TGM2 발현 억제를 통하여 미토콘드리아-소포체간 연접을 저해하여 소포체 칼슘의 미토콘드리아 내부로의 유입을 차단함을 규명하였으며, 이를 통해 장내미생물 대사물질인 유로리틴 A를 당뇨성 알츠하이머병의 예방 및 치료물질로 활용할 수 있다.In the present invention, it was found that urolithin A inhibits the mitochondrial-endoplasmic reticulum junction by inhibiting TGM2 expression, thereby blocking the influx of endoplasmic reticulum calcium into the mitochondria. Through this, urolitin A, an intestinal microbial metabolite, is used to treat diabetic Alzheimer's disease. It can be used as a preventive and therapeutic agent.
도 1 내지 도 7은 높은 포도당 조건에서 신경 세포의 미토콘드리아 칼슘, ROS에 대한 유로리틴의 효과에 관한 것이다.
도 8 내지 도 18은 높은 포도당 조건에서 신경 세포의 아밀로이드 생성에 대한 유로리틴 A의 효과에 관한 것이다.
도 19 내지 도 29는 높은 포도당 조건에서 MAM 조절 미토콘드리아 칼슘에서 유로리틴 A의 효과에 관한 것이다.
도 30 내지 도 34는 높은 포도당 유도 MAM 형성 및 아밀로이드 생성에서 TGM2의 역할에 관한 것이다.
도 35 내지 도 42는 AIP-AhR 전사 복합체 형성을 통한 TGM2 발현 억제에 대한 유로리틴 A의 효과에 관한 것이다.
도 43 내지 도 48은 Aβ에 의한 미토콘드리아 칼슘 유입, mtROS 축적 및 신경 세포 사멸에 대한 유로리틴 A의 보호 효과에 관한 것이다.
도 49 내지 도 54는 Aβ 유도 미토콘드리아 칼슘 과부하, mtROS 축적, 타우 인산화 및 신경 세포 사멸에서 TGM2의 역할에 관한 것이다.Figures 1 to 7 relate to the effects of urolithin on mitochondrial calcium and ROS in neurons under high glucose conditions.
Figures 8-18 relate to the effect of urolithin A on amyloid production in neurons under high glucose conditions.
Figures 19-29 relate to the effect of urolithin A on MAM regulating mitochondrial calcium under high glucose conditions.
Figures 30-34 relate to the role of TGM2 in high glucose-induced MAM formation and amyloidogenesis.
Figures 35-42 relate to the effect of urolitin A on suppressing TGM2 expression through AIP-AhR transcription complex formation.
Figures 43-48 relate to the protective effects of urolithin A against Aβ-induced mitochondrial calcium influx, mtROS accumulation and neuronal cell death.
Figures 49-54 relate to the role of TGM2 in Aβ-induced mitochondrial calcium overload, mtROS accumulation, tau phosphorylation and neuronal cell death.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.
본 발명의 일 양태는, 유로리틴 A를 유효성분으로 포함하는, 알츠하이머병의 예방 또는 치료용 약학 조성물을 제공한다. One aspect of the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising urolithin A as an active ingredient.
본 발명의 다른 하나의 양태는, 유로리틴 A를 유효성분으로 포함하는, 알츠하이머병의 예방 또는 개선용 건강기능식품 조성물을 제공한다.Another aspect of the present invention provides a health functional food composition for preventing or improving Alzheimer's disease, comprising urolithin A as an active ingredient.
본 발명의 다른 하나의 양태는, 후보물질 또는 유로리틴 A를 개체에 처리하는 단계; 개체의 미토콘드리아 활성산소종, LDH 생성, TGM2 발현량 또는 미토콘드리아 칼슘 축적의 정도를 비교하는 단계를 포함하는, 알츠하이머병의 예방 또는 치료 후보 물질을 스크리닝하는 방법을 제공한다.Another aspect of the present invention includes treating a subject with a candidate substance or urolithin A; Provided is a method for screening candidates for the prevention or treatment of Alzheimer's disease, comprising comparing the degree of mitochondrial reactive oxygen species, LDH production, TGM2 expression, or mitochondrial calcium accumulation of the subject.
본 발명에 있어서, 상기 알츠하이머병은 당뇨성 알츠하이머병인 것일 수 있다.In the present invention, the Alzheimer's disease may be diabetic Alzheimer's disease.
본 발명에 있어서, 상기 조성물은 고혈당 또는 고포도당 조건으로 유도된 미토콘드리아 활성산소종의 발생을 감소시키는 것이거나, 상기 조성물은 고혈당 또는 고포도당 조건으로 유도된 미토콘드리아 칼슘 축적을 억제하거나, 아밀로이드 베타의 생성 및 뉴런 변성을 억제하거나, 고혈당 또는 고포도당 조건으로 유도된 LDH의 생성을 낮추거나, TGM2 발현을 억제하거나, 및/또는 AIP-AhR 복합체의 형성을 촉진하는 것일 수 있다.In the present invention, the composition reduces the generation of mitochondrial reactive oxygen species induced by high blood sugar or high glucose conditions, the composition inhibits mitochondrial calcium accumulation induced by high blood sugar or high glucose conditions, or the production of amyloid beta. and suppressing neuronal degeneration, lowering the production of LDH induced by high blood sugar or high glucose conditions, suppressing TGM2 expression, and/or promoting the formation of the AIP-AhR complex.
본 발명에서 용어 “예방”이란 본 발명에 따른 조성물의 투여로 질환의 발병을 억제 또는 지연시키는 모든 행위를 말한다. 본 발명에서 용어 “치료”는 본 발명에 따른 조성물의 투여로 상기 질환의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다. 본 발명의 용어, “개선”이란, 본 발명의 조성물의 투여로 상기 질환이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.In the present invention, the term “prevention” refers to all actions that suppress or delay the onset of a disease by administering the composition according to the present invention. In the present invention, the term “treatment” refers to any action that improves or beneficially changes the symptoms of the disease by administering the composition according to the present invention. The term “improvement” of the present invention refers to any action in which the disease is improved or beneficially changed by administration of the composition of the present invention.
본 발명에서 사용된 용어, "투여"는 어떠한 적절한 방법으로 개체에 본 발명의 약학적 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다.As used in the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention into an individual by any appropriate method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through various routes.
본 발명에서 사용되는 용어 '개체' 알츠하이머 질환이 이미 발병하였거나 발병할 수 있는 인간을 포함한 모든 동물을 의미하고, 본 발명의 조성물을 개체에게 투여함으로써, 상기 질환을 효과적으로 예방 및 치료할 수 있다.The term 'individual' used in the present invention refers to all animals, including humans, that have already developed or may develop Alzheimer's disease, and that the disease can be effectively prevented and treated by administering the composition of the present invention to the subject.
본 발명의 다른 하나의 양태는, in vitro에서 유로리틴 A를 고포도당 조건의 세포에 처리하여 세포의 LDH의 생성을 낮추거나, 미토콘드리아 활성산소종의 발생을 감소시키거나, 미토콘드리아 칼슘 축적을 억제하거나, 아밀로이드 베타의 발현량을 감소시키거나, TGM2 발현을 억제하거나, 및/또는 AIP-AhR 복합체의 형성을 촉진하는 세포 처리 방법을 제공한다.Another aspect of the present invention is to treat cells under high glucose conditions with urolithin A in vitro to lower the production of cellular LDH, reduce the generation of mitochondrial reactive oxygen species, inhibit mitochondrial calcium accumulation, or , it provides a cell treatment method that reduces the expression level of amyloid beta, inhibits TGM2 expression, and/or promotes the formation of the AIP-AhR complex.
본 발명의 다른 하나의 양태는, 당뇨성 알츠하이머병 모델 마우스에 유로리틴 A를 처리하는 단계를 포함하는, 모델 마우스의 처리 방법으로서, Another aspect of the present invention is a method of treating a model mouse, comprising treating a diabetic Alzheimer's disease model mouse with urolithin A,
유로리틴 A를 고혈당 조건의 모델 마우스에 처리 또는 투여하여 마우스의 LDH의 생성을 낮추거나, 미토콘드리아 활성산소종의 발생을 감소시키거나, 미토콘드리아 칼슘 축적을 억제하거나, 아밀로이드 베타의 발현량을 감소시키거나, TGM2 발현을 억제하거나, 및/또는 AIP-AhR 복합체의 형성을 촉진하는 방법을 제공한다.Urolithin A is treated or administered to model mice under hyperglycemic conditions to lower the production of LDH in mice, reduce the generation of mitochondrial reactive oxygen species, inhibit mitochondrial calcium accumulation, or reduce the expression level of amyloid beta. , inhibiting TGM2 expression, and/or promoting the formation of the AIP-AhR complex.
본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용된 용어 '약학적으로 유효한 양'은 의학적 치료에 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 바이러스 감염 질환의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. The term 'pharmaceutically effective amount' used in the present invention refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the type and severity of the individual, age, It can be determined based on factors including gender, type of viral infectious disease, activity of the drug, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.
본 발명에 있어서, 상기 조성물은 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 추가로 포함할 수 있다.In the present invention, the composition may further include appropriate carriers, excipients, and diluents commonly used in manufacturing.
상기 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 이에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸셀룰로오스, 미정질셀룰로스, 폴리비닐피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods, respectively. Possible carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, not determined. Vaginal cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil.
상기 조성물을 제제화할 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제 적어도 면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트탈크 같은 윤활제들도 사용된다. 상기 약학적 조성물의 경구투여를 위한, 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세롤젤라틴 등이 사용될 수 있다.When formulating the composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations are made by mixing the above compound with at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration of the pharmaceutical composition include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, Sweeteners, flavoring agents, preservatives, etc. may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, Withepsol, Macrogol, Tween 61, cacao, laurin, glycerol gelatin, etc. can be used.
본 발명에 있어서, 상기 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 조성물은 1일 0.01 mg/kg 내지 10 g/kg으로, 바람직하게는 1 mg/kg 내지 1 g/kg으로 투여하는 것이 좋다. 투여는 하루에 한 번 투여할 수도 있고, 수회 나누어 투여할 수 있다.In the present invention, the preferred dosage of the composition varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, the composition is preferably administered at 0.01 mg/kg to 10 g/kg per day, preferably at 1 mg/kg to 1 g/kg. Administration may be administered once a day, or may be administered in several divided doses.
상기 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 통상의 방법에 의할 수 있고, 일 예로 경구 및 직장 또는 정맥등의 방법을 통하여 투여 할 수 있다.The composition can be administered to mammals such as rats, mice, livestock, and humans through various routes. All methods of administration may be conventional methods, for example, oral, rectal or intravenous administration.
본 발명에서 사용된 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 기능성이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능성 식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조 시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어날 수 있다.The term “health functional food” used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, functionality means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.
본 발명의 조성물을 건강기능식품에 포함하여 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 건강기능식품 또는 건강기능식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품의 제조 시에 본 발명의 조성물은 원료 조성물 중 1 ~ 10 중량%, 구체적으로 5 ~ 10 중량%의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하로도 사용될 수 있다.When using the composition of the present invention in a health functional food, the composition can be added as is or used together with other health functional foods or health functional food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food, the composition of the present invention is added in an amount of 1 to 10% by weight, specifically 5 to 10% by weight, of the raw material composition. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be used even below the above range.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 실험 재료 및 방법Example 1: Experimental Materials and Methods
1-1. 실험 재료1-1. experiment material
신경 모세포종 세포주 SH-SY5Y는 한국 세포주 은행에서 구매하였다. 아밀로이드 전구체 단백질 (APPSwe)의 스웨디시 돌연변이 (K595N / M596L)를 안정적으로 발현하는 SK-N-MC 신경 모세포종 세포주는 박기원 박사(연세대학교)로부터 제공받았으며, FBS, 유로리틴 A, 유로리틴 B, 유로리틴C, 유로리틴 D, 엘라그산, D-글루코스, 과산화수소, 시안화 카르보닐 m-클로로페닐하이드라존, Ru360, 안티마이신 A 등의 물질을 구매하여 준비하였다.Neuroblastoma cell line SH-SY5Y was purchased from the Korean Cell Line Bank. The SK-N-MC neuroblastoma cell line stably expressing the Swedish mutation (K595N/M596L) of amyloid precursor protein (APPSwe) was provided by Dr. Ki-Won Park (Yonsei University) and was supplied with FBS, urolithin A, urolithin B, and urolithin. Materials such as ritin C, urolithin D, ellagic acid, D-glucose, hydrogen peroxide, carbonyl cyanide m-chlorophenylhydrazone, Ru360, and antimycin A were purchased and prepared.
1-2. 세포 배양1-2. cell culture
SH-SY5Y 세포는 고포도당의 Dulbecco's Modified Eagle Medium (DMEM)에서 배양되었으며, 여기에 1 % 페니실린-스트렙토마이신 용액 (Gibco, Grand Island, NY, USA) 및 10 % FBS (Hyclone, Logan)를 보충하였다. APPSwe SK-N-MC 세포는 1 % 페니실린-스트렙토마이신 용액과 10% FBS가 포함된 고포도당 DMEM에서 배양하였다. 세포는 인큐베이터 (37 °C, CO2 5 % 및 공기 95 %)에서 60mm 접시, 100mm 배양 접시 또는 96-웰 플레이트에서 성장시켰다. 세포가 80 % confluency에 도달하면 배양 배지를 기아를 위해 24 시간 동안 무혈청 저-글루코오스 DMEM으로 교체하였다. 인큐베이션 후, 세포를 지정된 처리 시간 동안 지시된 제제로 보충된 무혈청 저-글루코오스 DMEM에서 인큐베이션 하였다. 높은 포도당 조건을 생성하기 위해 세포를 25mM D-글루코오스로 처리하였다.SH-SY5Y cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin-streptomycin solution (Gibco, Grand Island, NY, USA) and 10% FBS (Hyclone, Logan). . APPSwe SK-N-MC cells were cultured in high-glucose DMEM containing 1% penicillin-streptomycin solution and 10% FBS. Cells were grown in 60 mm dishes, 100 mm culture dishes, or 96-well plates in an incubator (37 °C, 5% CO 2 and 95% air). When cells reached 80% confluency, the culture medium was replaced with serum-free low-glucose DMEM for 24 h for starvation. After incubation, cells were incubated in serum-free low-glucose DMEM supplemented with the indicated agents for the indicated treatment times. To create high glucose conditions, cells were treated with 25mM D-glucose.
1-3. iPSC로부터의 신경 분화1-3. Neural differentiation from iPSCs
iPSC는 강스템 바이오텍(한국, 서울)에서 입수하였다. iPSC는 재조합 인간 비트로넥틴 (rhVTN, A14700, Thermo Fisher) 코팅 플레이트에서 배양하였다. 신경 줄기 세포 유도를 위해 PSC 신경 유도 배지 (A1647801, Thermo Fisher)를 사용하였다. iPSC에서 뉴런 분화 후, 라미닌-(23017, Thermo Fisher) 및 폴리-L-오르니틴 (Sigma, P3655) 코팅 접시에 신경 줄기 세포 (NSC)를 도포하였다. 신경 분화를 위해 NSC를 무혈청 보충제 B27 (17504, Thermo Fisher) 및 GlutaMax (35050, Thermo Fisher)와 함께 Neurobasal 배지 (21103, Thermo Fisher)에서 10 일 동안 배양했다. 신경 분화를 향상시키기 위해, 디부티릴-cAMP (D0627, Sigma)를 7 일과 10 일 사이에 신경 분화 배지에 첨가 하였다.iPSCs were obtained from Kangstem Biotech (Seoul, Korea). iPSCs were cultured on recombinant human vitronectin (rhVTN, A14700, Thermo Fisher) coated plates. PSC neural induction medium (A1647801, Thermo Fisher) was used for neural stem cell induction. After neuronal differentiation from iPSCs, neural stem cells (NSCs) were spread on laminin-(23017, Thermo Fisher) and poly-L-ornithine (Sigma, P3655) coated dishes. For neural differentiation, NSCs were cultured in Neurobasal medium (21103, Thermo Fisher) with serum-free supplements B27 (17504, Thermo Fisher) and GlutaMax (35050, Thermo Fisher) for 10 days. To enhance neural differentiation, dibutyryl-cAMP (D0627, Sigma) was added to the neural differentiation medium between days 7 and 10.
1-4. LDH 세포 독성 분석1-4. LDH cytotoxicity assay
LDH 방출 분석 키트 (EZ-LDH, DoGenBio, Seoul, Korea, DG-LDH500)에 제공된 프로토콜에 따라 세포 농도를 최적화했다. SH-SY5Y 세포는 96-웰 플레이트에 1 Х 104 세포 / 웰의 밀도로 접종되었다. 세포가 90 % confluency에 도달하면 배지를 무혈청 저 글루코오스 DMEM으로 교체했다. 600Хg에서 플레이트를 원심분리 한 후, 상청액을 수집하고 LDH 분석 혼합물과 함께 실온 (RT)에서 30 분 동안 배양하였다. LDH 방출 수준은 Epoch 2 분광 광도계 (BioTek, VT, USA)를 사용하여 450 nm에서 광학 밀도를 측정하여 분석했다.Cell concentration was optimized according to the protocol provided in the LDH release assay kit (EZ-LDH, DoGenBio, Seoul, Korea, DG-LDH500). SH-SY5Y cells were seeded in 96-well plates at a density of 1 Х 10 4 cells/well. When cells reached 90% confluency, the medium was replaced with serum-free, low-glucose DMEM. After centrifugation of the plate at 600Хg, the supernatant was collected and incubated with the LDH assay mixture for 30 minutes at room temperature (RT). LDH emission levels were analyzed by measuring optical density at 450 nm using an Epoch 2 spectrophotometer (BioTek, VT, USA).
1-5. 수용성 테트라졸륨염 (WST-1) 세포 생존력 분석1-5. Water-soluble tetrazolium salt (WST-1) cell viability assay
SH-SY5Y 세포의 생존력은 WST-1 세포 생존력 분석 (EZ-Cytox ; Daeil Labservice, Seoul, Korea, # EZ-1000)으로 측정되었다. 세포를 96-well plate에서 80 % confluency까지 배양한 후, 무혈청 저포도당 DMEM으로 성장 배지를 교체하였다. 유로리틴 A (100 nM) 또는 과산화수소 (100 μM)를 세포에 30 분 동안 첨가한 후 72 시간 동안 D-포도당을 처리했다. 세포는 37 ℃에서 30 분 동안 100 μl의 DMEM에있는 10 μl의 EZ-Cytox 시약에서 배양되었다. 450 nm에서의 흡광도는 Epoch 2 분광 광도계로 측정되었다.The viability of SH-SY5Y cells was measured by WST-1 cell viability assay (EZ-Cytox; Daeil Labservice, Seoul, Korea, #EZ-1000). After culturing the cells in a 96-well plate until 80% confluency, the growth medium was replaced with serum-free, low-glucose DMEM. Urolithin A (100 nM) or hydrogen peroxide (100 μM) was added to cells for 30 min and then treated with D-glucose for 72 h. Cells were incubated in 10 μl of EZ-Cytox reagent in 100 μl of DMEM for 30 min at 37 °C. Absorbance at 450 nm was measured with an Epoch 2 spectrophotometer.
1-6. mtROS 및 칼슘 측정 및 미토콘드리아 투과성 전이 기공 분석1-6. mtROS and calcium measurements and mitochondrial permeability transition pore analysis
MitoSOX Red (Thermo Fisher, M36008) 및 rhod-2 (Thermo Fisher, R1244)를 사용하여 세포 내 ROS, mtROS 및 미토콘드리아 칼슘 수준을 측정했다. mPTP 개방 수준을 Calcein AM (Thermo Fisher, C1430)을 사용하여 분석하여 mPTP 개방을 측정하여 세포 사멸을 결정했다. CoCl2 (400 μM)를 첨가하여 세포질 칼슘을 급냉시켰다. 각 실험은 Cytoflex 유세포 분석기 (Beckman Coulter, FL, USA)를 사용하여 수행되었으며, 결과는 높은 형광 강도를 가진 세포의 비율을 비교하여 얻었다.Intracellular ROS, mtROS and mitochondrial calcium levels were measured using MitoSOX Red (Thermo Fisher, M36008) and rhod-2 (Thermo Fisher, R1244). The level of mPTP opening was analyzed using Calcein AM (Thermo Fisher, C1430) to determine cell death by measuring mPTP opening. Cytosolic calcium was quenched by adding CoCl 2 (400 μM). Each experiment was performed using a Cytoflex flow cytometer (Beckman Coulter, FL, USA), and results were obtained by comparing the proportion of cells with high fluorescence intensity.
1-7. 동물 연구의 실험 설계1-7. Experimental design of animal studies
동물 연구 프로토콜은 서울대학교 위원회(SNU-190801-4-1)의 승인을 받아 진행하였다. 9 주령 수컷 CrljOri : CD1 (ICR) 마우스는 Orient-Bio에서 수득하였다. 각 마우스의 무게를 측정 한 후 22 ° C, 70 % 상대 습도, 12 시간 빛 : 어두운주기의 통제된 무균 조건에서 3 일 동안 수용하고 정상적인 식단과 물을 무제한 공급하였다. STZ는 0.05M 시트 레이트 완충액에 용해되었고, 마우스는 STZ (75mg / kg / day) 또는 시트레이트 완충액 (0.05M)을 총 부피 200μl로 3 일 동안 복강 주사했다. 모든 마우스에게 7 일 동안 10 % 수크로스 물을 제공하여 STZ 주사된 마우스에서 저포도당 쇼크의 가능성을 낮추었다. 꼬리에서 혈액을 채취하고 혈당 측정기 (Accu-Chek, Roche Diagnostics, Indianapolis, IN)를 사용하여 STZ 주입 7 일 후 혈당 수치를 측정했다. 유로리틴 A를 PBS 중의 0.5mM NaOH에 용해시켰다. 200μl 부피의 유로리틴 A (2.5mg / kg / 일) 또는 0.5mM NaOH의 매일 복강 내 주사를 8 주 동안 투여했다. 마우스는 모든 실험 동안 하루에 두 번 모니터링되었다. 8 주간의 유로리틴 A 또는 비히클 처리 후 각 마우스의 체중을 측정하고 꼬리 혈액의 혈당치를 측정하였다.The animal research protocol was approved by the Seoul National University Committee (SNU-190801-4-1). Nine-week-old male CrljOri:CD1 (ICR) mice were obtained from Orient-Bio. Each mouse was weighed and housed for 3 days under controlled germ-free conditions at 22°C, 70% relative humidity, 12 h light:dark cycle and provided with a normal diet and water ad libitum. STZ was dissolved in 0.05 M citrate buffer, and mice were intraperitoneally injected with STZ (75 mg/kg/day) or citrate buffer (0.05 M) in a total volume of 200 μl for 3 days. All mice were provided with 10% sucrose water for 7 days to reduce the likelihood of low-glucose shock in STZ-injected mice. Blood was collected from the tail and blood glucose levels were measured 7 days after STZ injection using a glucometer (Accu-Chek, Roche Diagnostics, Indianapolis, IN). Urolithin A was dissolved in 0.5mM NaOH in PBS. Daily intraperitoneal injections of urolithin A (2.5 mg/kg/day) or 0.5 mM NaOH in a volume of 200 μl were administered for 8 weeks. Mice were monitored twice daily during all experiments. After 8 weeks of urolithin A or vehicle treatment, each mouse was weighed and the blood glucose level of tail blood was measured.
1-8. Y- 미로 자발적 교대 테스트1-8. Y-maze voluntary alternation test
STZ 유발 당뇨 마우스와 정상 마우스는 8 주 동안 유로리틴 A (2.5mg / kg / day) 또는 비히클을 주사했다. Y-미로 자발적 교대 테스트 전에 마우스를 3 시간 동안 테스트 룸에 수용하여 외부 환경 자극 또는 의도하지 않은 스트레스가 행동에 미치는 영향을 줄였다. 먼저, 마우스를 Y-미로 장치에 위치시켰다. 다음으로 마우스에게 8 분 동안 Y-미로를 탐색 할 기회를 부여하고 비디오 카메라를 사용하여 마우스의 움직임을 기록했다. STZ-induced diabetic mice and normal mice were injected with urolithin A (2.5 mg/kg/day) or vehicle for 8 weeks. Mice were housed in the test room for 3 h before the Y-maze voluntary alternation test to reduce the influence of external environmental stimuli or unintentional stress on behavior. First, the mouse was placed in the Y-maze apparatus. Next, mice were given the opportunity to explore the Y-maze for 8 min, and their movements were recorded using a video camera.
1-9. Transglutaminase 2 활성 측정1-9. Transglutaminase 2 activity measurement
Transglutaminase (TGM) 2 활성 분석 키트는 탈아미드화 활성으로부터 하이드록사메이트 생산물의 생산에 적용된다. 하이드록사메이트는 최종적으로 정지 용액과 함께 보라색 복합체를 형성하고 광학 흡광도는 마이크로플레이트 리더로 525 nm에서 측정되었다. TGM2 활성은 세포 용해물 샘플의 총 단백질에 의해 표준화되었다.Transglutaminase (TGM) 2 activity assay kit is applied to the production of hydroxamate product from deamidation activity. Hydroxamate finally formed a purple complex with the stop solution, and the optical absorbance was measured at 525 nm with a microplate reader. TGM2 activity was normalized by total protein in cell lysate samples.
1-10. siRNA 형질 감염1-10. siRNA transfection
SH-SY5Y 세포는 항생제 없이 24 시간 동안 표시된 siRNA 및 형질 감염 시약 TurboFect (Thermo Fisher, R0531)의 25 nM과 함께 배양되었다. 배지를 무혈청 저글루코오스 DMEM으로 변경하였다. TGM2에 대한 실시간 qPCR에 의한 siRNA 효능이 70 % 이상임을 확인하였으며, 대조군으로 NT siRNA를 사용 하였다.SH-SY5Y cells were incubated with 25 nM of the indicated siRNA and transfection reagent TurboFect (Thermo Fisher, R0531) for 24 h without antibiotics. The medium was changed to serum-free low glucose DMEM. The siRNA efficacy was confirmed to be over 70% by real-time qPCR for TGM2, and NT siRNA was used as a control.
1-11. 실시간 정량 PCR1-11. Real-time quantitative PCR
세포는 고포도당 또는 비히클로 24 시간 동안 처리되었다. 이어서 세포를 PBS로 2회 세척하고 50X 디티오트레이톨 용액을 함유하는 완충액 RL로 용해시켰다. RNA 추출 키트 (Takara, Japan, 9767)를 사용하여 제조업체의 지침에 따라 총 RNA를 추출했다. 역전사 PCR은 Maxime RT premix kit (iNtRON, Sungnam, Korea, 25081)를 사용하여 1 μg의 total RNA로 수행하였다. cDNA는 Maxime PCR PreMix Kit (iNtRON, 25165) 및 MyGenie 96 (Bioneer, 대전, 대한민국)을 사용하여 증폭되었다. 표적 유전자의 상대적 mRNA 발현 수준은 TB Green Premix Ex Taq (TaKaRa, RR420A)와 함께 Rotor-Gene 6000 장치 (Corbett Research, Cambridge, UK)를 사용하여 분석되었다. PCR 산물을 확인하고 용융 곡선을 분석하여 실시간 정량 PCR 용 PCR 프라이머의 특이성, 효율성 및 충실도를 검증했다. VDAC1, MCU1, MICU1, MICU2, MCUR1, MCUB, BAX, BCL2L1, BCL2, GRP75, TGM2, PTGES3, AIP 및 HSP90AA1의 상대적 mRNA 발현 수준은 delta-delta Ct 방법으로 분석되었다. 18s rRNA는 데이터 정규화를 위한 참조 유전자로 사용되었다.Cells were treated with high glucose or vehicle for 24 h. Cells were then washed twice with PBS and lysed with buffer RL containing 50X dithiothreitol solution. Total RNA was extracted using an RNA extraction kit (Takara, Japan, 9767) according to the manufacturer's instructions. Reverse transcription PCR was performed with 1 μg of total RNA using the Maxime RT premix kit (iNtRON, Sungnam, Korea, 25081). cDNA was amplified using the Maxime PCR PreMix Kit (iNtRON, 25165) and MyGenie 96 (Bioneer, Daejeon, South Korea). Relative mRNA expression levels of target genes were analyzed using a Rotor-Gene 6000 device (Corbett Research, Cambridge, UK) with TB Green Premix Ex Taq (TaKaRa, RR420A). PCR products were identified and melting curves were analyzed to verify the specificity, efficiency and fidelity of PCR primers for real-time quantitative PCR. The relative mRNA expression levels of VDAC1, MCU1, MICU1, MICU2, MCUR1, MCUB, BAX, BCL2L1, BCL2, GRP75, TGM2, PTGES3, AIP and HSP90AA1 were analyzed by delta-delta Ct method. 18s rRNA was used as a reference gene for data normalization.
1-12. 웨스턴 블로팅 및 세포내 분획1-12. Western blotting and subcellular fractionation
단백질 농도는 비신코닌산 (BCA) 단백질 분석 키트 (Pierce, Rockford, IL, USA, 23225)에 의해 결정하였다. 샘플 단백질은 SDS-PAGE 겔 전기영동에 의해 분해되고 PVDF 막으로 옮겼다. 막은 4 °C에서 밤새 1 차 항체와 함께 배양되었다. 특정 밴드는 ChemiDoc XRS + System (Bio-Rad, Richmond, CA, USA)에 의해 시각화되었다. 세포질 및 핵 단백질을 분리하기 위해 세포내 분획을 수행하였다. 세포를 100mm 접시에서 배양하고 표시된 시약으로 처리했다. 세포질 및 핵 분획 샘플의 제조를 위해 EzSubcell 세포내 분획 / 추출 키트 (Atto, Tokyo, Japan, WSE-7421)를 사용했다. 웨스턴 블롯 분석을 위한 세포질 및 핵 샘플은 제조업체의 지침에 따라 준비되었다. α-tubulin 및 lamin A / C는 각각 세포질 및 핵 단백질 마커로 사용되었다.Protein concentration was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA, 23225). Sample proteins were resolved by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Membranes were incubated with primary antibodies overnight at 4 °C. Specific bands were visualized by ChemiDoc XRS + System (Bio-Rad, Richmond, CA, USA). Subcellular fractionation was performed to separate cytoplasmic and nuclear proteins. Cells were cultured in 100 mm dishes and treated with the indicated reagents. The EzSubcell subcellular fractionation/extraction kit (Atto, Tokyo, Japan, WSE-7421) was used for preparation of cytoplasmic and nuclear fraction samples. Cytoplasmic and nuclear samples for Western blot analysis were prepared according to the manufacturer's instructions. α-tubulin and lamin A/C were used as cytoplasmic and nuclear protein markers, respectively.
1-13. Aβ 분비를 위한 엘라이사 (ELISA)1-13. ELISA for Aβ secretion
세포 배양 배지에서 Aβ (142)의 정량화를 위해 공급 업체의 프로토콜에 따라 Aβ 42 Human ELISA (Thermo Fisher, KHB3544)를 진행하였다. SH-SY5Y 세포는 80 % confluency까지 성장하도록 배양되었으며, 상청액을 수집하고 ELISA를 수행했다. 전두엽 피질 및 해마의 마우스 뇌 조직 샘플을 수집하고 RIPA 완충액 (ATTO, Tokyo, Japan)으로 용해시켰다. Aβ 42 마우스 ELISA (Thermo Fisher, KMB3441)는 공급 업체의 지시에 따라 수행하였다.For quantification of Aβ(142) in cell culture medium, Aβ 42 Human ELISA (Thermo Fisher, KHB3544) was performed according to the supplier's protocol. SH-SY5Y cells were cultured to grow to 80% confluency, supernatants were collected and ELISA was performed. Mouse brain tissue samples from the prefrontal cortex and hippocampus were collected and lysed with RIPA buffer (ATTO, Tokyo, Japan). Aβ 42 mouse ELISA (Thermo Fisher, KMB3441) was performed according to the supplier's instructions.
1-14. 면역 세포 화학1-14. immunocytochemistry
면역 세포 화학을 위해 SH-SY5Y 세포를 4 % 파라포름알데히드로 10 분 동안 고정한 다음 0.5 % Tween-20에서 10 분 동안 배양했다. 세포를 0.1 % Tween-20 (PBST; 1 : 100 희석)을 함유하는 PBS에서 1 차 항체와 함께 2 시간 동안 배양하고 PBS로 3 회 세척하였다. 세포를 PBST (1 : 100 희석)에서 Alexa Fluor 488 또는 555- 접합 이차 항체와 함께 1 시간 동안 배양했다. 면역 형광 염색된 샘플은 초고해상도 방사상 변동 (SRRF) 이미징 시스템 (Andor Technology, Belfast, UK)에 의해 시각화되었다. AhR / DAPI의 상대적 형광 강도는 ImageJ 소프트웨어로 정량화되었다. ER-미토콘드리아 접촉을 분석하기 위해 세포를 화학 물질로 처리하거나 TGM2 siRNA로 형질 감염시키고 24 시간 동안 배양했다. 세포를 PBS로 3 회 세척 한 후 세포를 혈청이 없는 배지에서 37 °C에서 20 분 동안 MitoTracker green (200nM) 및 ER-Tracker (200nM)와 함께 배양하고 핵을 Hoechst 33342 (Thermo Fisher)로 염색했다. For immunocytochemistry, SH-SY5Y cells were fixed with 4% paraformaldehyde for 10 min and then incubated in 0.5% Tween-20 for 10 min. Cells were incubated with primary antibodies in PBS containing 0.1% Tween-20 (PBST; 1:100 dilution) for 2 h and washed three times with PBS. Cells were incubated with Alexa Fluor 488 or 555-conjugated secondary antibodies in PBST (1:100 dilution) for 1 h. Immunofluorescently stained samples were visualized by a super-resolution radial fluctuation (SRRF) imaging system (Andor Technology, Belfast, UK). The relative fluorescence intensity of AhR/DAPI was quantified with ImageJ software. To analyze ER-mitochondrial contacts, cells were treated with chemicals or transfected with TGM2 siRNA and cultured for 24 h. After washing the cells three times with PBS, cells were incubated with MitoTracker green (200 nM) and ER-Tracker (200 nM) for 20 min at 37 °C in serum-free medium, and nuclei were stained with Hoechst 33342 (Thermo Fisher). .
1-15. in situ PLA1-15. in situ PLA
VDAC1 / IP3R1 상호 작용은 공급 업체의 프로토콜에 따라 Duolink II 2 차 항체 및 검출 키트 (Sigma-Aldrich, DUO92001, DUO92005 및 DUO92008)를 사용하여 in situ로 검출하였다. 세포를 고정하고 PLA 프로브 항-VDAC1 및 항-IP3R1 항체를 적용한 후 이차 항체가 추가되었다. 항체가 매우 근접해 있으면 (<40nm) 서로 연결되게 된다. 중합 및 증폭 용액을 보충하여 닫힌 원의 신호 (빨간색)를 증폭하고 SRRF 현미경으로 시각화했다. DAPI는 핵을 대조 염색하는데 사용되었다. VDAC1/IP3R1 interaction was detected in situ using Duolink II secondary antibodies and detection kits (Sigma-Aldrich, DUO92001, DUO92005 and DUO92008) according to the supplier's protocol. Cells were fixed and PLA probe anti-VDAC1 and anti-IP3R1 antibodies were applied followed by the addition of secondary antibodies. When antibodies are in close proximity (<40 nm), they become linked to each other. The signal in the closed circle (red) was amplified by supplementing the polymerization and amplification solution and visualized by SRRF microscopy. DAPI was used to counterstain nuclei.
1-16. 공동 면역 침전1-16. Co-immunoprecipitation
NT 또는 TGM2 siRNA로 형질 감염된 SH-SY5Y 세포를 비히클 또는 D-글루코스 (25mM)로 24 시간 동안 처리한 다음, 얼음 위에서 30 분 동안 프로테아제 억제제 칵테일을 포함하는 공동 면역 침전 용해 완충액 (20mM Tris-HCl pH 8.0, 137mM NaCl, 1 % Nonidet P- 40, 및 2mM EDTA)으로 용해하였다. 각 용해물에서 단백질의 농도는 BCA 정량 분석 (Thermo Fisher, 23225)에 의해 결정되었다. VDAC1 또는 토끼 IgG 항체는 단백질 G 자기 비드 (Sure Beads, Bio-Rad, CA, USA, 161-4021)로 고정되었다. 고정된 자기 비드는 4 °C에서 6 시간 동안 세포 용해물과 함께 배양되었다. 세척된 비드는 20mM 글리신 완충액 (pH 2.0)으로 5 분 동안 용리되고 1M 인산염 완충액 및 라엠리 샘플 완충액으로 중화되었다. 이후 단백질 샘플을 100 °C에서 5 분 동안 가열하였다.SH-SY5Y cells transfected with NT or TGM2 siRNA were treated with vehicle or D-glucose (25mM) for 24 h and then incubated in co-immunoprecipitation lysis buffer (20mM Tris-HCl pH) containing protease inhibitor cocktail for 30 min on ice. 8.0, 137mM NaCl, 1% Nonidet P-40, and 2mM EDTA). The concentration of protein in each lysate was determined by BCA quantitative analysis (Thermo Fisher, 23225). VDAC1 or rabbit IgG antibodies were immobilized with protein G magnetic beads (Sure Beads, Bio-Rad, CA, USA, 161-4021). The immobilized magnetic beads were incubated with cell lysates for 6 h at 4 °C. Washed beads were eluted with 20mM glycine buffer (pH 2.0) for 5 min and neutralized with 1M phosphate buffer and Laemli sample buffer. The protein sample was then heated at 100 °C for 5 min.
1-17. 통계 분석1-17. statistical analysis
모든 정량 데이터는 평균 ± 평균의 표준 오차로 표시되었다. 데이터는 SigmaPlot 12 소프트웨어를 사용하여 분석되었고, 동물 연구를위한 샘플 크기는 SigmaPlot 12 소프트웨어에 의해 결정되었다. 두 실험군 간의 비교는 two-tailed Student's t test을 사용하여 수행되었다. 여러 실험 그룹의 평균은 일원 분산 분석을 사용하여 비교한 다음 다중 비교를 위해 Student-Newman-Keuls 테스트를 사용했다. <0.05의 p 값은 통계적으로 유의한 것으로 간주되었다.All quantitative data were expressed as mean ± standard error of the mean. Data were analyzed using SigmaPlot 12 software, and sample sizes for animal studies were determined by SigmaPlot 12 software. Comparison between the two experimental groups was performed using the two-tailed Student's t test. The means of multiple experimental groups were compared using one-way analysis of variance, followed by the Student-Newman-Keuls test for multiple comparisons. A p value of <0.05 was considered statistically significant.
실시예 2: 실험 결과Example 2: Experimental results
2-1. 고포도당 유도 미토콘드리아 칼슘 유입 및 mtROS 축적에 대한 유로리틴 A의 효과2-1. Effect of urolithin A on high glucose-induced mitochondrial calcium influx and mtROS accumulation.
먼저, 고혈당 조건에서 SH-SY5Y 세포의 세포 생존력에 대한 엘라그산과 유로리틴의 효과를 조사했다. 유로리틴 A, 유로리틴 B, 유로리틴 C, 유로리틴 D의 전처리는 높은 포도당 유도 LDH 방출 수준을 유의하게 감소시켰다 (도 1). 고혈당군의 유로리틴 A 전처리에서 LDH 방출 수준이 다른 전처리에 비해 가장 낮았다. WST-1 세포 생존력 분석에서 높은 포도당을 가진 유로리틴 A 전처리된 SHSY5Y 세포의 생존율이 높은 포도당을 가진 비히클 전처리된 SH-SY5Y 세포보다 더 높은 것을 확인했다. MitoSOX 염색 분석은 유로리틴 A 전처리가 높은 포도당 유도 mtROS 수준을 감소시키는 것으로 나타났다 (도 2). 그러나 엘라그산과 다른 유로리틴의 효과가 유의하지 않은 것으로 나타났다. 또한, 24 ~ 48 시간 동안 높은 포도당 상태는 SH-SY5Y에서 rhod-2-양성 세포 집단을 증가시켰으며, 이는 높은 포도당이 미토콘드리아 칼슘 유입을 유도함을 나타낸다 (도 3). 유로리틴 A 전처리가 높은 포도당 자극 미토콘드리아 칼슘 축적을 억제하지만 미토콘드리아 투과성 전이 기공 (mPTP) 개방을 억제한다는 것을 발견했다 (도 4, 5). 높은 포도당 유도 세포내 및 mtROS 수준은 MCU (미토콘드리아 칼슘 유니포터) 억제제 Ru360 (도 6, 7)으로 전처리하여 억제되었다. 이러한 결과는 미토콘드리아 칼슘 축적 감소에 대한 유로리틴 A의 효과가 높은 포도당 하에서 SH-SY5Y 세포에서 mtROS 항상성을 유지하는 데 중요하다는 것을 나타낸다.First, we investigated the effects of ellagic acid and urolithin on cell viability of SH-SY5Y cells under hyperglycemic conditions. Pretreatment with urolitin A, urolitin B, urolitin C, and urolitin D significantly reduced the level of high glucose-induced LDH release (Figure 1). In the hyperglycemia group, the LDH release level was lowest in the urolithin A pretreatment compared to other pretreatments. In the WST-1 cell viability assay, it was confirmed that the survival rate of SHSY5Y cells pretreated with urolithin A with high glucose was higher than that of SH-SY5Y cells pretreated with vehicle with high glucose. MitoSOX staining analysis showed that urolithin A pretreatment reduced high glucose-induced mtROS levels (Figure 2). However, the effects of ellagic acid and other urolithins were not found to be significant. Additionally, high glucose conditions for 24 to 48 h increased the rhod-2-positive cell population in SH-SY5Y, indicating that high glucose induces mitochondrial calcium influx (Figure 3). We found that urolithin A pretreatment inhibited high glucose-stimulated mitochondrial calcium accumulation but not mitochondrial permeability transition pore (mPTP) opening (Figures 4, 5). High glucose-induced intracellular and mtROS levels were suppressed by pretreatment with the MCU (mitochondrial calcium uniporter) inhibitor Ru360 (Figures 6, 7). These results indicate that the effect of urolithin A on reducing mitochondrial calcium accumulation is important for maintaining mtROS homeostasis in SH-SY5Y cells under high glucose.
2-2. 유로리틴 A가 신경 세포 및 스트렙토조토신 (STZ) 유발 당뇨병 생쥐에서 높은 포도당 유도 아밀로이드 생성 및 신경 변성에 미치는 영향2-2. Effects of urolithin A on high glucose-induced amyloidogenesis and neurodegeneration in neurons and streptozotocin (STZ)-induced diabetic mice.
고혈당 하에서 뉴런 세포의 아밀로이드 생성에 대한 유로리틴의 효과를 확인하기 위해 SH-SY5Y 및 iPSC 유래 신경 분화 세포 (iPSC-ND)에서 분비된 Aβ (1-42) 수준을 측정했다. 높은 농도의 포도당이 시간에 따라 APP 및 BACE1 발현을 증가시키는 것을 발견했다. 또한, 유로리틴 A와 유로리틴 B를 포함하지만 엘라그산, 유로리틴 C, 유로리틴 D가 아닌 배지에서 배양한 SH-SY5Y 세포에서 Aβ (1-42) 농도 수준이 현저하게 감소하는 것을 확인했다 (도 8). Aβ 분비에 대한 유로리틴 A의 억제 효과는 다른 기질에 비해 가장 강력했기 때문에 고혈당 하에서 iPSC-ND 세포의 Aβ 농도에 대한 유로리틴 A의 효과를 테스트했다. iPSCND 컨디셔닝 배지에서 고혈당 군을 사용한 유로리틴 A 전처리의 Aβ (1-42) 농도는 고혈당 군을 사용한 비히클 전처리보다 낮았다 (도 9). 유로리틴 A 전처리는 SH-SY5Y 및 iPSC-ND 세포 모두에서 높은 포도당 유도 APP 및 BACE1 발현을 억제했다 (도 10). 높은 포도당 유도 APP 및 BACE1 발현 수준은 Ru360 또는 mtROS- 특이적 스캐빈저 Mito-TEMPO 전처리에 의해 억제되었다 (도 11, 12). 이러한 결과는 유로리틴 A-억제 Aβ 생산이 미토콘드리아 칼슘 및 mtROS 축적 억제에 의해 매개됨을 시사한다. 또한, STZ 유발 인지 장애, 마우스 뇌 조직의 전두엽 피질과 해마에서 아밀로이드 생성, Tau 인산화 및 Aβ 침착에 대한 유로리틴 A의 효과를 결정했다. 유로리틴 A는 대조군 또는 STZ 마우스에서 체중 증가에 영향을 미치지 않았다 (도 13). 유로리틴 A 주사 대조군 마우스의 혈당 수준은 비히클 주사 대조군 마우스보다 유의하게 낮았지만, 비히클 주사 STZ와 유로리틴 A 주사 STZ 마우스 간의 이러한 차이는 통계적으로 유의하지 않았다 (도 14). 인지 기능을 평가하기 위해 Y-maze 자발적 교대 테스트를 수행했다. STZ 주입이 유로리틴 A 주입에 의해 역전된 자발적 교대 속도를 감소시키는 것을 관찰했다 (도 15). 전두엽 피질과 해마 조직에서 유로리틴 A 주사는 STZ 유도 당뇨병 마우스의 APP, BACE1, p-Tau (S262 및 S396)와 Aβ 수준 (1-42)을 억제했다 (도 16~18). 따라서, 시험관내 및 생체내 실험 결과는 유로리틴 A 치료가 DM 모델에서 아밀로이드 생성 및 뉴런 변성을 억제할 수 있음을 시사한다.To determine the effect of urolithin on amyloid production in neuronal cells under high glucose, we measured the levels of secreted Aβ(1-42) in SH-SY5Y and iPSC-derived neural differentiated cells (iPSC-ND). We found that high concentrations of glucose increased APP and BACE1 expression in a time-dependent manner. In addition, we confirmed that Aβ (1-42) concentration levels were significantly reduced in SH-SY5Y cells cultured in medium containing urolithin A and urolithin B but not ellagic acid, urolithin C, or urolithin D ( Figure 8). Since the inhibitory effect of urolithin A on Aβ secretion was the strongest compared to other substrates, we tested the effect of urolithin A on Aβ concentration in iPSC-ND cells under high glucose. In iPSCND conditioned medium, the Aβ(1-42) concentration of urolithin A pretreatment with high glucose group was lower than vehicle pretreatment with high glucose group (Figure 9). Urolithin A pretreatment inhibited high glucose-induced APP and BACE1 expression in both SH-SY5Y and iPSC-ND cells (Figure 10). High glucose-induced APP and BACE1 expression levels were suppressed by pretreatment with Ru360 or the mtROS-specific scavenger Mito-TEMPO (Figures 11, 12). These results suggest that urolithin A-inhibited Aβ production is mediated by inhibition of mitochondrial calcium and mtROS accumulation. Additionally, the effects of urolithin A on STZ-induced cognitive impairment, amyloidogenesis, Tau phosphorylation, and Aβ deposition in the prefrontal cortex and hippocampus of mouse brain tissue were determined. Urolithin A had no effect on body weight gain in control or STZ mice (Figure 13). Blood glucose levels in urolithin A-injected control mice were significantly lower than vehicle-injected control mice, but this difference between vehicle-injected STZ and urolithin A-injected STZ mice was not statistically significant (Figure 14). The Y-maze voluntary alternation test was performed to assess cognitive function. We observed that STZ infusion reduced the rate of spontaneous alternation, which was reversed by urolithin A infusion (Figure 15). In prefrontal cortex and hippocampus tissue, urolithin A injection suppressed APP, BACE1, p-Tau (S262 and S396) and Aβ levels (1-42) in STZ-induced diabetic mice (Figures 16-18). Therefore, the in vitro and in vivo experimental results suggest that urolithin A treatment can inhibit amyloid production and neuronal degeneration in the DM model.
2-3. 트랜스글루타미나 제2형 (TGM2)의 역할은 높은 포도당 하에서 미토콘드리아-ER 접촉, 미토콘드리아 칼슘 유입, mtROS 축적 및 아밀로이드 생성 감소에 대한 유로리틴 A의 효과.2-3. Role of transglutaminase type 2 (TGM2) Effects of urolithin A on reducing mitochondrial-ER contacts, mitochondrial calcium influx, mtROS accumulation, and amyloidogenesis under high glucose.
VDAC1, MCU1, 미토콘드리아 칼슘 섭취 1 (MICU1), 미토콘드리아 칼슘 섭취 2 (MICU2), MCU 조절자 1 (MCUR1) 및 MCU-우성 음성 베타 서브 유닛 (MCUB)와 같이 미토콘드리아 칼슘 유입을 조절하는 단백질의 mRNA 발현에 대한 고혈당의 영향을 조사했다. 우리는 VDAC1과 MCU1의 mRNA 발현 수준이 높은 포도당 조건 하에서 SH-SY5Y에서 상향 조절된다는 것을 발견했다 (도 19). 그러나, 유로리틴 A 전처리는 높은 포도당 유도 VDAC1 및 MCU1 mRNA와 단백질 발현에 영향을 미치지 않았다 (도 20, 21). 또한 Ru360 전처리는 높은 포도당 자극 VDAC1 및 MCU1 단백질 발현을 변경하지 않았다. 본 발명자들은 ER과 미토콘드리아 간의 상호 작용에 대한 높은 포도당과 유로리틴 A의 효과를 추가로 조사했다. 도 22에서 볼 수 있듯이, 높은 포도당이 SH-SY5Y 세포에서 MitoTracker 양성 형광과 ER-Tracker 양성 형광의 공동 국소화를 증가시켰으며, 이는 유로리틴 A 전처리에 의해 반전되었다. coimmunoprecipitation 및 in situ 근접 결찰 분석 (PLA) 실험에서 높은 포도당은 IP3R1, IP3R3 및 VDAC1 간의 직접적인 상호 작용을 증가시켰으며, 이는 유로리틴 A 전처리에 의해 억제되었다 (도 23, 24, 25). 또한 Bcl-2-연관 X 단백질 (BAX), Bcl-2-like1 (BCL2L1), B 세포 림프종 2 (BCL2), 포도당-조절된 단백질 75 (GRP75) 및 TGM2과 같은 미토콘드리아-ER 접촉 조절 어댑터 단백질의 mRNA 발현 변화를 조사했다. 그러나 높은 포도당은 TGM2 mRNA 발현을 자극할 뿐 유로리틴 A 전처리에 의해 역전되었다 (도 26). 높은 포도당에 의한 TGM2 유도는 MitoTEMPO 전처리에 의해 억제되었다. SH-SY5Y와 iPSC-ND 세포 모두에서 높은 포도당 유도 TGM2 발현에 대한 유로리틴 A 전처리의 억제 효과를 확인했다 (도 27). 유로리틴 A inhibited TGM2 발현 외에도 SH-SY5Y 세포에서 높은 포도당 유도 TGM2 활성을 감소시켰다 (도 28). 마우스 뇌 샘플에서 유로리틴 A 주사는 전두엽 피질과 해마 조직 모두에서 STZ- 유도 TGM2 발현을 유의하게 감소시켰다 (도 29). 종합하면, 유로리틴 A는 높은 포도당 환경에 노출된 신경 세포에서 IP3R1-VDAC1 상호 작용을 억제하는 데 중요한 TGM2 발현을 억제했다. 높은 포도당 하에서 미토콘드리아-ER 접촉과 아밀로이드 생성에서 TGM2의 역할을 결정하기 위해, SH-SY5Y 세포에서 TGM2 silencing 효과를 조사했다. TGM2 침묵이 SH-SY5Y 세포에서 MitoTracker 양성 형광과 ER-Tracker 양성 형광의 높은 포도당 유도 공동 국소화를 저해했음을 관찰했다 (도 30). 공동 면역 침전과 PLA 실험에서 TGM2 침묵은 높은 포도당 유도 IP3R1-VDAC1 상호 작용을 하향 조절했다 (도 31, 32). SH-SY5Y 세포에서 높은 포도당 유도 미토콘드리아 칼슘과 mtROS 수준은 TGM2 침묵에 의해 억제되었다 (도 33). 또한, TGM2 침묵은 높은 포도당 유도 Aβ 분비뿐만 아니라 APP 및 BACE1 발현을 방지했다 (도 34). 이러한 발견에 기초하여, 유로리틴 비정규화된 고포도당 유도 TGM2 발현은 IP3R1-VDAC1 상호 작용을 방해하여 고포도당 하에서 미토콘드리아 칼슘 유입 및 mtROS 축적을 억제함을 알 수 있다.mRNA expression of proteins that regulate mitochondrial calcium influx, such as VDAC1, MCU1, mitochondrial calcium uptake 1 (MICU1), mitochondrial calcium uptake 2 (MICU2), MCU regulator 1 (MCUR1) and MCU-dominant negative beta subunit (MCUB) The effect of hyperglycemia on was investigated. We found that the mRNA expression levels of VDAC1 and MCU1 were upregulated in SH-SY5Y under high glucose conditions (Figure 19). However, urolithin A pretreatment had no effect on high glucose-induced VDAC1 and MCU1 mRNA and protein expression (Figures 20, 21). Additionally, Ru360 pretreatment did not alter high glucose-stimulated VDAC1 and MCU1 protein expression. We further investigated the effects of high glucose and urolithin A on the interaction between ER and mitochondria. As shown in Figure 22, high glucose increased co-localization of MitoTracker-positive and ER-Tracker-positive fluorescence in SH-SY5Y cells, which was reversed by urolithin A pretreatment. In coimmunoprecipitation and in situ proximity ligation assay (PLA) experiments, high glucose increased the direct interaction between IP3R1, IP3R3, and VDAC1, which was inhibited by urolithin A pretreatment (Figures 23, 24, 25). Additionally, the expression of mitochondria-ER contact regulatory adapter proteins such as Bcl-2-associated Changes in mRNA expression were investigated. However, high glucose only stimulated TGM2 mRNA expression, which was reversed by urolithin A pretreatment (Figure 26). TGM2 induction by high glucose was inhibited by MitoTEMPO pretreatment. We confirmed the inhibitory effect of urolithin A pretreatment on high glucose-induced TGM2 expression in both SH-SY5Y and iPSC-ND cells (Figure 27). In addition to urolithin A inhibited TGM2 expression, it also reduced high glucose-induced TGM2 activity in SH-SY5Y cells (Figure 28). In mouse brain samples, urolithin A injection significantly reduced STZ-induced TGM2 expression in both prefrontal cortex and hippocampal tissue (Figure 29). Taken together, urolithin A inhibited TGM2 expression, which is important for inhibiting IP3R1-VDAC1 interaction in neurons exposed to a high glucose environment. To determine the role of TGM2 in mitochondria-ER contacts and amyloidogenesis under high glucose, we examined the effect of TGM2 silencing in SH-SY5Y cells. We observed that TGM2 silencing inhibited high glucose-induced colocalization of MitoTracker-positive and ER-Tracker-positive fluorescence in SH-SY5Y cells (Figure 30). In co-immunoprecipitation and PLA experiments, TGM2 silencing downregulated high glucose-induced IP3R1-VDAC1 interaction (Figs 31, 32). High glucose-induced mitochondrial calcium and mtROS levels in SH-SY5Y cells were suppressed by TGM2 silencing (Figure 33). Additionally, TGM2 silencing prevented high glucose-induced Aβ secretion as well as APP and BACE1 expression (Figure 34). Based on these findings, urolithin-denormalized high-glucose-induced TGM2 expression disrupts the IP3R1-VDAC1 interaction, inhibiting mitochondrial calcium influx and mtROS accumulation under high glucose.
2-4. 고포도당 유도된 TGM2 발현에서 유로리틴 A 매개 AIP-AhR 복합체 촉진 의 역할2-4. Role of urolithin A-mediated AIP-AhR complex promotion in high-glucose-induced TGM2 expression.
유로리틴 A가 높은 포도당 유발 TGM2 발현을 억제하는 방법의 메커니즘을 확인하고자 하였다. 아릴 탄화수소 수용체 (AhR)의 단백질 발현 수준이 고혈당 처리 24 시간과 48 시간에서 증가했지만 AhR-상호 단백질 (AIP)은 증가하지 않은 것을 확인하였다 (도 35). 이후, 고농도의 포도당 세포 SH-SY5Y에서 prostaglandin E synthase 3 (PTGES3), AIP 및 열충격 단백질 90 α family class A member 1 (HSP90AA1)과 같은 AhR 조절 어댑터 단백질의 mRNA 발현에 대한 유로리틴 A의 효과를 확인하였다. 그러나 높은 포도당과 유로리틴 A는 SHSY5Y 세포에서 AIP의 mRNA 또는 단백질 발현에 영향을 미치지 않았다. 높은 포도당이 핵 AhR 발현 수준을 증가시키며, 이는 유로리틴 A 전처리에 의해 억제되었다 (도 36, 37). 높은 포도당 유도 TGM2 mRNA와 단백질 발현 수준은 AhR 억제제 CH-223191로 전처리함으로써 억제되었다 (도 38, 39). 또한, 높은 포도당은 유로리틴 A 전처리에 의해 회복된 AhR과 AIP 사이의 상호 작용을 유의하게 억제했다 (도 40, 41, 42). 이러한 발견은 유로리틴 A가 TGM2 발현을 억제하기 위해 AIP-AhR 복합체의 형성을 촉진함으로써 높은 포도당 활성화 AhR 신호를 억제한다는 것을 나타낸다.We sought to determine the mechanism of how urolithin A suppresses high glucose-induced TGM2 expression. It was confirmed that the protein expression level of aryl hydrocarbon receptor (AhR) increased at 24 and 48 hours of high glucose treatment, but that of AhR-interacting protein (AIP) did not increase (FIG. 35). Afterwards, we confirmed the effect of urolitin A on the mRNA expression of AhR-regulated adapter proteins such as prostaglandin E synthase 3 (PTGES3), AIP, and heat shock protein 90 α family class A member 1 (HSP90AA1) in SH-SY5Y high-glucose cells. did. However, high glucose and urolithin A did not affect the mRNA or protein expression of AIP in SHSY5Y cells. High glucose increased nuclear AhR expression levels, which was suppressed by urolithin A pretreatment (Figures 36, 37). High glucose-induced TGM2 mRNA and protein expression levels were suppressed by pretreatment with the AhR inhibitor CH-223191 (Figures 38, 39). Additionally, high glucose significantly inhibited the interaction between AhR and AIP, which was restored by urolithin A pretreatment (Figures 40, 41, 42). These findings indicate that urolithin A inhibits high glucose-activated AhR signaling by promoting the formation of the AIP-AhR complex to suppress TGM2 expression.
2-5. Aβ 자극 미토콘드리아 칼슘 유입, mtROS 축적, Tau 인산화 및 신경 세포 사멸에 대한 유로리틴 A 및 TGM2 침묵의 보호 효과 2-5. Protective effects of urolithin A and TGM2 silencing on Aβ -stimulated mitochondrial calcium influx, mtROS accumulation, Tau phosphorylation, and neuronal apoptosis.
다음으로, Aβ 자극 미토콘드리아 칼슘 유입, mtROS 축적 및 신경 세포 사멸에 대한 유로리틴 A의 효과를 조사했다. Aβ 처리는 48 시간과 72 시간에 SH-SY5Y 세포에서 LDH 방출 수준을 유의하게 증가시켰다 (도 43). SH-SY5Y 및 iPSC-ND 세포에서 유로리틴 A 전처리는 Aβ 증가 미토콘드리아 칼슘 수치를 억제했다 (도 44, 45). 유로리틴 A 전처리가 SHSY5Y와 iPSC-ND 세포에서 Aβ에 의한 mtROS 축적을 막는 것을 확인했다 (도 46, 47). 더욱이, 유로리틴 A 전처리는 72 시간 동안 Aβ로 처리 된 SHSY5Y 세포로부터 LDH 방출을 막았다 (도 48). 본 발명자들은 Aβ 처리된 신경 세포 및 Aβ를 분비하는 APP 스웨디시 돌연변이 SK-N-MC 세포 (APPSwe)에 대한 TGM2 침묵의 효과를 추가로 조사했다. TGM2 침묵은 SH-SY5Y 세포에서 Aβ감소된 미토콘드리아 칼슘 수준을 감소시켰다 (도 49). TGM2 침묵 또는 유로리틴 A 전처리는 APPSwe 세포에서 Aβ 증가된 미토콘드리아 칼슘 수치를 감소 시켰다 (도 50). 또한, TGM2 침묵은 SH-SY5Y에서 Aβ유도 mtROS 축적을 억제했으며 (도 51) TGM2 siRNA로 형질 감염된 APPSwe 세포는 NT siRNA로 형질 감염된 APPSwe 세포보다 낮은 mtROS 수준을 나타냈다 (도 52). 유로리틴 A 전처리는 또한 APPSwe 세포에서 Aβ에 의한 mtROS 축적을 억제했다(도 52). TGM2 침묵화는 Aβ자극된 Tau 인산화를 상당하게 탈인산화하였다 (도 53). APPSwe 세포에서 TGM2 침묵과 유로리틴 A 전처리는 모두 Tau 인산화를 감소시켰다 (도 54). Aβ 처리를 한 SH-SY5Y 세포의 LDH 방출 수준도 TGM2 침묵에 의해 감소되었다 (도 55). 종합하면, 상기 실시예의 결과는 유로리틴 A 전처리 및 TGM2 침묵이 Aβ 유도 미토콘드리아 칼슘 유입, mtROS 축적, Tau 인산화 및 신경 세포에서 세포 사멸을 방지한다는 것을 알 수 있다.Next, we investigated the effects of urolithin A on Aβ-stimulated mitochondrial calcium influx, mtROS accumulation and neuronal cell death. Aβ treatment significantly increased the level of LDH release in SH-SY5Y cells at 48 and 72 h (Figure 43). In SH-SY5Y and iPSC-ND cells, urolithin A pretreatment inhibited Aβ increased mitochondrial calcium levels (Figures 44, 45). It was confirmed that urolithin A pretreatment prevented Aβ-induced mtROS accumulation in SHSY5Y and iPSC-ND cells (Figures 46 and 47). Moreover, urolithin A pretreatment blocked LDH release from SHSY5Y cells treated with Aβ for 72 h (Figure 48). We further investigated the effect of TGM2 silencing on Aβ-treated neurons and APP Swedish mutant SK-N-MC cells (APPSwe) secreting Aβ. TGM2 silencing reduced Aβ reduced mitochondrial calcium levels in SH-SY5Y cells (Figure 49). TGM2 silencing or urolithin A pretreatment reduced Aβ increased mitochondrial calcium levels in APPSwe cells (Figure 50). Additionally, TGM2 silencing suppressed Aβ-induced mtROS accumulation in SH-SY5Y (Figure 51) and APPSwe cells transfected with TGM2 siRNA showed lower mtROS levels than APPSwe cells transfected with NT siRNA (Figure 52). Urolithin A pretreatment also inhibited Aβ-induced mtROS accumulation in APPSwe cells (Figure 52). TGM2 silencing significantly dephosphorylated Aβ-stimulated Tau phosphorylation (Figure 53). In APPSwe cells, both TGM2 silencing and urolithin A pretreatment reduced Tau phosphorylation (Figure 54). The level of LDH release from SH-SY5Y cells treated with Aβ was also reduced by TGM2 silencing (Figure 55). Taken together, the results of the above examples show that urolithin A pretreatment and TGM2 silencing prevent Aβ-induced mitochondrial calcium influx, mtROS accumulation, Tau phosphorylation, and cell death in neuronal cells.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.
Claims (6)
In in vitro conditions, treatment of neuroblastoma cell lines SH-SY5 or iPSC-ND under high glucose conditions with urolithin A reduced LDH production in these cells; Reduce mitochondrial reactive oxygen species generation; Inhibition of mitochondrial calcium accumulation; Reduced expression of amyloid beta; Inhibition of TGM2 expression; and promoting the formation of an AIP-AhR complex.
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