KR20220085164A - Composition for inhibiting adipocyte differentiation comprising a mussel protein-derived peptide as active ingredient - Google Patents
Composition for inhibiting adipocyte differentiation comprising a mussel protein-derived peptide as active ingredient Download PDFInfo
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- KR20220085164A KR20220085164A KR1020200175004A KR20200175004A KR20220085164A KR 20220085164 A KR20220085164 A KR 20220085164A KR 1020200175004 A KR1020200175004 A KR 1020200175004A KR 20200175004 A KR20200175004 A KR 20200175004A KR 20220085164 A KR20220085164 A KR 20220085164A
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- adipocyte differentiation
- peptide
- expression
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Abstract
본 발명은 홍합단백질로부터 분리한 펩타이드 서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는지방세포분화 억제용 조성물 및 비만개선용 조성물을 개시한다. heme oxygenase-1(HO-1)의 발현을 증가시킴으로써 지방세포분화를 억제 및 지방분해를 촉진하는 홍합단백질 유래 펩타이드를 함유하는 지방세포분화 억제용 조성물 및 지방분해 촉진용 조성물을 개시하고 있다. 본 발명에 따르면 홍합단백질 유래 펩타이드가 heme oxygenase-1(HO-1)의 발현시켜 지방세포분화에 관여하는 전자인자의 발현을 억제하며 지방세포에서 지방분해를 촉진하여 세포내 지방의 과잉 축적을 억제할 수 있어 지방세포분화 억제 및 비만개선의 효과를 얻을 수 있다. The present invention discloses a composition for inhibiting adipocyte differentiation and a composition for improving obesity, comprising, as an active ingredient, a peptide comprising the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK), isolated from mussel protein. Disclosed are a composition for inhibiting adipocyte differentiation and a composition for promoting lipolysis containing a mussel protein-derived peptide that inhibits adipocyte differentiation and promotes lipolysis by increasing the expression of heme oxygenase-1 (HO-1). According to the present invention, the mussel protein-derived peptide suppresses the expression of electron factors involved in adipocyte differentiation by expressing heme oxygenase-1 (HO-1), and promotes lipolysis in adipocytes to suppress excessive accumulation of intracellular fat. Therefore, it is possible to obtain the effect of suppressing the differentiation of adipocytes and improving obesity.
Description
본 발명은 서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 지방세포분화 억제용 조성물 및 지방분해 촉진 조성물에 관한 것으로, 보다 상세하게는 heme oxygenase-1(HO-1)의 발현을 증가시킴으로써 지방세포분화를 억제 및 지방분해를 촉진하는 홍합단백질 유래 서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 지방세포분화 억제용 조성물 및 지방분해 촉진 조성물에 관한 것이다The present invention relates to a composition for inhibiting adipocyte differentiation and a composition for promoting lipolysis comprising, as an active ingredient, a peptide comprising the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK), and more particularly, heme oxygenase- 1 (HO-1) containing, as an active ingredient, a peptide comprising the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK) derived from mussel protein that inhibits adipocyte differentiation and promotes lipolysis by increasing the expression of To a composition for inhibiting adipocyte differentiation and a composition for promoting lipolysis
비만은 음식물의 섭취(food intake)와 에너지의 사용(energy expenditure)의 불균형으로 초래되는 질병으로, 염증, 제2형 당뇨병 및 동맥경화 등 심각한 질병을 동반할 수 있다. 최근 대사증후군(metabolic syndrome)으로 부르게 되었으며, 이런 성인병들은 사회적으로 가장 왕성하게 활동하는 사람들에게서 발병하기 때문에 경제적인 손실도 상당할 뿐만 아니라, 이의 예방과 치료를 위해 들이는 비용은 의약학 및 건강 산업의 최고액에 다다를 정도의 수준이다.Obesity is a disease caused by an imbalance between food intake and energy expenditure, and may accompany serious diseases such as inflammation,
종래부터 비만의 예방과 개선에 대해서는 식사제한, 과잉 에너지의 흡수 지연 및 저해, 혹은 소화관에서의 당질흡수 저해물질의 탐색 등 섭취 에너지를 제한하기 위한 다양한 연구가 이루어져 왔다. 그러나, 에너지 섭취의 제한은 기초대사량을 저하시키는 요인도 되므로, 비만이 개선되지 않는 경우도 있다. 따라서, 비만을 해소하기 위해서는 축적된 지방을 적극적으로 대사, 분해하여 열에너지로서 발산하는 것이 이상적이다. 이에, 근래 식품소재 중에서 지방분해 촉진작용을 갖는 기능성 성분의 탐색이 활발하게 이루어져 많은 지방분해촉진제 및 음식품이 제안되고 있다.Conventionally, for the prevention and improvement of obesity, various studies have been made to limit energy intake, such as diet restriction, delay and inhibition of absorption of excess energy, or search for substances that inhibit carbohydrate absorption in the digestive tract. However, restriction of energy intake is also a factor that lowers the basal metabolic rate, so there are cases in which obesity does not improve. Therefore, in order to solve obesity, it is ideal to actively metabolize and decompose accumulated fat and dissipate it as heat energy. Accordingly, in recent years, the search for functional ingredients having a lipolysis promoting action among food materials has been actively made, and many lipolysis promoting agents and food and drink have been proposed.
항비만 치료제 개발은 주로 당질의 소화에 관여하는 아밀라아제나 α-글루코시다제에 대한 저해기능을 갖는 소화흡수 억제제, 식욕억제제, 및 체내에 축적된 체지방의 대사를 조절하기 위한 지방대체제, 지방대사 개선제, 및 혈당 감소제를 기반으로 한 저분자 합성 화합물이 주를 이루고 있다.The development of anti-obesity treatment agents mainly has an inhibitory function on amylase and α-glucosidase, which are involved in the digestion of carbohydrates, an appetite suppressant, and a fat substitute for regulating the metabolism of body fat accumulated in the body, a fat metabolism improving agent , and low-molecular-weight synthetic compounds based on blood sugar reducing agents are predominant.
이들 치료제들은 대부분이 중추신경계에 작용하여 식욕을 억제시켜는 약물로써 경구투여의 이점이 있지만 미미한 체중 감소효과, 요요현상 및 향정신성 의약품으로 남용 가능성에 주의가 필요한 단점이 있다. Most of these treatments are drugs that suppress appetite by acting on the central nervous system and have advantages of oral administration.
지방조직의 분화는 근육세포 분화나 신경세포 분화와는 달리 여러 호르몬과 다양한 전사인자들의 상호작용을 통하여 매우 복잡하게 이루어진다. 지방전구세포가 지방세포로 분화해 나가는 과정에는 세포의 형태적 변화와 유전자 발현양상의 변화 등이 함께 일어난다. 대부분의 경우 이러한 변화는 전사단계에서 각 유전자의 발현양의 변화를 나타내는데, 지방세포의 분화조절은 C/EBP, PPAR, r/RXR, ADD1/SREBP 1C라고 불리는 전사인자(transcription factor)가 중추적인 역할을 담당하고 있다. 이들 전사인자는 지방세포 분화 과정 중 각기 다른 시점에서 발현이 유도되며 서로 상호작용을 통하여 여러 지방세포 특이유전자들의 발현을 조절하고, 지방대사의 활성화와 지방세포 분화를 점진적으로 유도해 나간다.Differentiation of adipose tissue is very complicated through the interaction of various hormones and various transcription factors, unlike muscle cell differentiation or nerve cell differentiation. In the process of differentiation of preadipocytes into adipocytes, cell morphological changes and gene expression changes occur together. In most cases, these changes indicate changes in the expression level of each gene at the transcriptional stage, and the regulation of adipocyte differentiation is driven by transcription factors called C/EBP, PPAR, r/RXR, and ADD1/SREBP 1C. is playing a role. Expression of these transcription factors is induced at different points in the adipocyte differentiation process, and through interaction with each other, the expression of various adipocyte-specific genes is controlled, and fat metabolism is activated and adipocyte differentiation is gradually induced.
최근에는 기존 치료제의 부작용을 회피할 수 있는 펩타이드를 이용한 제재개발이 활발히 진행되고 있으며 다양한 분자 targets을 활용한 비만치료제가 개발되고 있다.Recently, drug development using peptides that can avoid the side effects of existing treatments is being actively developed, and anti-obesity drugs using various molecular targets are being developed.
상기 문제점을 해결하기 위하여, 본 발명은 지방세포분화에 관여하는 전자인자의 발현을 억제하며 지방세포에서 지방분해를 촉진하여 세포 내 지방의 과잉 축적을 억제하는 지방세포분화 억제용 조성물을 제공하는 것을 목적으로 한다.In order to solve the above problems, the present invention is to provide a composition for inhibiting adipocyte differentiation, which suppresses the expression of electronic factors involved in adipocyte differentiation and promotes lipolysis in adipocytes to suppress excessive accumulation of intracellular fat. The purpose.
또한, 본 발명은 홍합단백질로부터 유래의 펩타이드를 이용하여 heme oxygenase-1(HO-1)의 발현을 증가시킴으로써 지방세포분화를 억제하는 방법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a method for inhibiting adipocyte differentiation by increasing the expression of heme oxygenase-1 (HO-1) using a peptide derived from mussel protein.
또한, 본 발명은 홍합단백질로부터 유래의 펩타이드를 이용하여 heme oxygenase-1(HO-1)의 발현을 증가시킴으로써 지방분해 촉진용 조성물을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a composition for promoting lipolysis by increasing the expression of heme oxygenase-1 (HO-1) using a peptide derived from mussel protein.
상기 목적을 해결하기 위하여 본 발명은,The present invention in order to solve the above object,
서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 지방세포분화 억제 및 지방분해 촉진용 조성물을 제공한다.Provided is a composition for inhibiting adipocyte differentiation and promoting lipolysis comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK) as an active ingredient.
상기 펩타이드는 홍합단백질로부터 유래할 수 있다.The peptide may be derived from mussel protein.
상기 펩타이드에 의하여 heme oxygenase-1(HO-1)의 발현을 증가시키고 β-catenin의 활성화를 유도하여 지방세포분화를 억제할 수 있다.The peptide can inhibit adipocyte differentiation by increasing the expression of heme oxygenase-1 (HO-1) and inducing the activation of β-catenin.
상기 펩타이드는 전사인자인 PPARγ 및 C/EBPα를 억제하여 지방세포분화를 조절할 수 있다.The peptide can regulate adipocyte differentiation by inhibiting transcription factors PPARγ and C/EBPa.
상기 펩타이드는 p-AMPK를 활성화하여 지방분해를 촉진할 수 있다.The peptide may promote lipolysis by activating p-AMPK.
상기 다른 목적을 해결하기 위하여 본 발명은,The present invention in order to solve the other object,
서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 이용하는 지방세포분화 억제 및 지방분해 촉진 방법을 제공한다.Provided is a method for inhibiting adipocyte differentiation and promoting lipolysis using a peptide comprising the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK).
상기 다른 목적을 해결하기 위하여 본 발명은,The present invention in order to solve the other object,
서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 지방분해 촉진 조성물을 제공한다.It provides a composition for promoting lipolysis comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK) as an active ingredient.
본 발명은 홍합단백질 유래 펩타이드가 heme oxygenase-1(HO-1)의 발현을 증가시켜 지방세포분화에 관여하는 전자인자 억제 및 지방분해를 촉진하는 단백질 발현을 증가시켜 지방세포분화 억제 및 지방세포에서 지방분해를 촉진하여 세포내 지방의 과잉 축적을 억제할 수 있어 비만개선의 효과를 얻을 수 있다.In the present invention, the mussel protein-derived peptide increases the expression of heme oxygenase-1 (HO-1) to suppress the electronic factor involved in adipocyte differentiation and increase the expression of a protein that promotes lipolysis, thereby inhibiting adipocyte differentiation and improving adipocyte differentiation. By promoting lipolysis, excessive accumulation of intracellular fat can be suppressed, thereby obtaining the effect of improving obesity.
도 1은 본 발명의 일실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드가 중간엽줄기세포의 지방세포분화 시 지방 축적 및 지방 분해에 미치는 영향을 도시한 것이다.
도 2는 본 발명의 일 실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드가 중간엽줄기세포의 지방세포분화 시 전사인자 및 지방산 생합성 및 분해 효소들의 발현에 미치는 영향을 도시한 것이다.
도 3은 본 발명의 일 실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드 처리가 중간엽줄기세포의 지방세포분화 과정에서 염증성 사이토카인 및 활성산소종 생성에 미치는 영향을 도시한 것이다.
도 4는 본 발명의 일 실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드가 중간엽줄기세포의 지방세포분화 과정에서 HO-1 발현 및 전사인자 활성화에 미치는 영향을 도시한 것이다.
도 5는 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 지방구 형성 및 분해에 미치는 영향을 도시한 것이다.
도 6은 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 β-catenin 활성화에 미치는 영향을 도시한 것이다.
도 7은 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 활성산소종 및 염증성 사이토카인 생성에 미치는 영향을 도시한 것이다.
도 8은 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 HO-1 및 전사인자 단백질 발현에 미치는 영향을 도시한 것이다.
도 9는 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 AMPK 활성화에 미치는 영향을 도시한 것이다.1 shows the effect of PIISVYWK and FSVVPSPK peptides on fat accumulation and lipolysis during adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention.
Figure 2 shows the effect of PIISVYWK and FSVVPSPK peptides on the expression of transcription factors and fatty acid biosynthesis and degradation enzymes during adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention.
Figure 3 shows the effect of PIISVYWK and FSVVPSPK peptide treatment on the production of inflammatory cytokines and reactive oxygen species in the process of adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention.
Figure 4 shows the effect of PIISVYWK and FSVVPSPK peptides on HO-1 expression and transcription factor activation in the process of adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention.
5 shows the effect of HO-1 inhibitor treatment on adipocyte formation and degradation during adipocyte differentiation according to an embodiment of the present invention.
6 shows the effect of HO-1 inhibitor treatment on β-catenin activation in the process of adipocyte differentiation according to an embodiment of the present invention.
7 shows the effect of HO-1 inhibitor treatment on the production of reactive oxygen species and inflammatory cytokines in the process of adipocyte differentiation according to an embodiment of the present invention.
8 shows the effect of HO-1 inhibitor treatment on HO-1 and transcription factor protein expression during adipocyte differentiation according to an embodiment of the present invention.
9 shows the effect of HO-1 inhibitor treatment on AMPK activation during adipocyte differentiation according to an embodiment of the present invention.
본 명세서에 있어서, 어떤 부분이 어떤 구성요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다. 그리고 본 명세서에서 사용된 용어는 실시예들을 설명하기 위한 것이며, 본 발명을 제한하고자 하는 것이 아니다. In the present specification, when a part "includes" a certain component, this means that other components may be further included rather than excluding other components unless otherwise stated. And the terminology used in this specification is for describing the embodiments, and is not intended to limit the present invention.
이하 본 발명에 대하여 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일측면에 따르면, 서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 지방세포분화 억제 및 지방분해 촉진용 조성물을 제공한다.According to one aspect of the present invention, there is provided a composition for inhibiting adipocyte differentiation and promoting lipolysis, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK) as an active ingredient.
본 발명은 서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드는 홍합단백질로부터 형성될 수 있다.In the present invention, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK) can be formed from a mussel protein.
본 발명의 홍합단백질 유래 펩타이드에 의하여 heme oxygenase-1(HO-1)의 발현이 증가시키고 β-catenin의 활성화를 유도하여 지방세포분화를 억제할 수 있다.The mussel protein-derived peptide of the present invention can inhibit adipocyte differentiation by increasing the expression of heme oxygenase-1 (HO-1) and inducing activation of β-catenin.
본 발명의 홍합단백질 유래 펩타이드(PIISVYWK 및 FSVVPSPK)가 지방세포분화에 필수적인 전사인자(PPARγ, C/EBPα, SREBP1)의 발현에 영향을 미칠 수 있다. SREBP-1는 지방산 생합성 (fatty acid synthase, FAS; lipoprotein lipase, LPL) 효소의 발현에 관여하며 protein kinase A(PKA)에 의해서 지방산 분해에 관여하는 효소인 HSL(hormone sensitive lipase)의 활성화에 관여한다.The mussel protein-derived peptides (PIISVYWK and FSVVPSPK) of the present invention may affect the expression of transcription factors essential for adipocyte differentiation (PPARγ, C/EBPa, SREBP1). SREBP-1 is involved in the expression of fatty acid biosynthesis (fatty acid synthase, FAS; lipoprotein lipase, LPL) enzymes and is involved in the activation of hormone sensitive lipase (HSL), an enzyme involved in fatty acid degradation by protein kinase A (PKA). .
또한 본 발명의 펩타이드 처리를 하게 되면 지방세포분화 과정에서 생성되는 염증성 사이토카인 및 활성산소종을 효과적을 억제할 수 있다.In addition, treatment with the peptide of the present invention can effectively inhibit inflammatory cytokines and reactive oxygen species generated in the process of adipocyte differentiation.
HO-1(heme oxygenase-1)은 외부 자극에 의해서 발현되는 대표적인 세포보호 효소로서 전사인자인 Nrf2의 활성화 기작에 의해서 발현된다. HO-1 발현은 세포 내에서 항산화 및 항염증뿐만 아니라 비만 관련 대사증후군 및 심혈관질환을 감소시키는 것으로 확인된다. HO-1은 지방세포분화를 촉진하는 것으로 알려져 있는 heme 단백질을 분해시켜 지방세포분화를 억제할 수 있다. 또한 HO-1은 염증성 사이토카인등의 생성을 억제하여 염증 반응을 줄여주는 것으로도 알려져 있다.HO-1 (heme oxygenase-1) is a representative cytoprotective enzyme expressed by external stimuli, and is expressed by the activation mechanism of the transcription factor Nrf2. HO-1 expression has been shown to reduce anti-oxidation and anti-inflammatory properties in cells, as well as obesity-related metabolic syndrome and cardiovascular disease. HO-1 can inhibit adipocyte differentiation by degrading heme protein, which is known to promote adipocyte differentiation. In addition, HO-1 is also known to reduce the inflammatory response by inhibiting the production of inflammatory cytokines.
HO-1의 발현이 Wnt/β-catenin 신호전달에 관여하는 것으로 알려져 있으며, 활성화된 β-catenin 전사인자는 지방세포분화에 필수적인 전사인자인 PPARγ 및 C/EBPα를 억제하여 지방세포분화를 조절하는 것으로 알려져 있다. 그러므로 본 발명에 따라, 서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드 처리를 하면 HO-1 발현을 통하여 β-catenin의 활성화(핵이동) 및 HO-1 저해제 처리에 의한 β-catenin의 활성화에 영향을 미친다는 것이다.The expression of HO-1 is known to be involved in Wnt/β-catenin signaling, and the activated β-catenin transcription factor regulates adipocyte differentiation by inhibiting PPARγ and C/EBPa, transcription factors essential for adipocyte differentiation. it is known Therefore, according to the present invention, when a peptide comprising the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK) is treated, β-catenin activation (nuclear migration) and HO-1 inhibitor treatment through HO-1 expression It is said that it affects the activation of β-catenin by
본 발명에 따라 지방세포분화 과정에서 홍합단백질 유래 펩타이드(PIISVYWK 및 FSVVPSPK) 처리에 의한 HO-1의 발현은 Wnt/β-catenin의 활성화를 유도하며 이는 지방세포분화에 필수적인 전사인자를 조절하여 지방세포분화를 억제하는 것으로 볼 수 있다.According to the present invention, expression of HO-1 by treatment with mussel protein-derived peptides (PIISVYWK and FSVVPSPK) in the process of adipocyte differentiation induces the activation of Wnt/β-catenin, which regulates transcription factors essential for adipocyte differentiation and thus adipocytes. It can be seen to inhibit differentiation.
본 발명에 따라 지방세포분화 과정에서 홍합단백질 유래 펩타이드(PIISVYWK 및 FSVVPSPK) 처리에 의한 HO-1의 발현은 p-AMPK의 활성화를 유도하며 이는 지방분해를 촉진하여 지방세포에서 지질의 축적을 억제하는 것으로 볼 수 있다. 따라서 본 발명은 서열번호 1(PIISVYWK) 또는 서열번호 2(FSVVPSPK)의 아미노산 서열로 이루어진 펩타이드를 유효성분으로 포함하는 지방분해 촉진 조성물을 제공한다.According to the present invention, expression of HO-1 by treatment with mussel protein-derived peptides (PIISVYWK and FSVVPSPK) in the process of adipocyte differentiation induces the activation of p-AMPK, which promotes lipolysis and suppresses the accumulation of lipids in adipocytes. can be seen as Accordingly, the present invention provides a composition for promoting lipolysis comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK).
본 발명에 따른 펩타이드는 식품 유래 저분자 펩타이드 소재로 안전성이 확보되어 있으며 부작용이 없다는 장점이 있다.The peptide according to the present invention is a food-derived low-molecular-weight peptide material, and has the advantage of ensuring safety and having no side effects.
본 발명에 따른 지방세포분화 억제용 조성물은 지방세포분화를 억제하는 효과를 낼 수 있어 기존 비만치료제의 부작용을 회피할 수 있고 비만의 개선과 치료를 위한 다양한 형태의 약학 조성물로 활용이 가능하다.The composition for inhibiting adipocyte differentiation according to the present invention can have an effect of inhibiting adipocyte differentiation, thereby avoiding the side effects of existing anti-obesity drugs, and can be used as various types of pharmaceutical compositions for improvement and treatment of obesity.
본 발명의 약학 조성물은 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용 가능한 담체를 포함하는 상기 약학 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention may contain an acceptable carrier. The pharmaceutical composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablet pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose, or lactose. ), gelatin, etc. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
본 발명의 약학 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있다.The pharmaceutical composition of the present invention is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have any one formulation.
본 발명의 약학 조성물은 치료적 유효량 또는 약학으로 유효한 양으로 투여한다. 용어 "치료적 유효량 또는 약학으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a therapeutically effective amount or a pharmaceutically effective amount. The term "therapeutically effective amount or pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type and severity of the subject, age, sex, and activity of the drug. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field.
이하, 본 명세서를 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다. 그러나, 본 명세서에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 명세서의 범위가 아래에서 기술하는 실시예들에 한정되는 것으로 해석되지 않는다. 본 명세서의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 명세서를 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be given to describe the present specification in detail. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those of ordinary skill in the art.
<실시예><Example>
<실험재료 및 방법><Test materials and methods>
재료ingredient
홍합(Mytilus edulis) 단백질 유래 항산화 펩타이드 2종(PIISVYWK, 1004.57Da; FSVVPSPK, 860.09Da)은 Fmoc SPSS(solid phase peptide synthesis) 방법으로 합성하였다(Peptron Inc. 서울). 그 외 세포 배양에 사용된 재료는 Gibro-BRL(Gaithersburg, MD, USA)에서 구입하였다. 단백질 분석을 위한 항체(HO-1, Nrf2, β-catenin, PPARγ, SREBP-1, C/EBPα, LPL, FAS, HSL 및 β-actin)는 Santa Cruz Biotechnology(Santa Cruz, CA, USA)에서 구입하였고, 염증성 사이토카인 측정에 사용된 분석 kits는 GE Healthcare (Buckinghamshire, UK)에서 구입하였다. 항산화 효소(catalase, glutathione peroxidase 및 superoxide dismutases) 측정에 사용된 분석 kits는 Cayman 社(MI, USA)에서 구입하였고, 그 외 시약은 Sigma-Aldrich 社에서 구입하였다.Two types of mussel ( Mytilus edulis ) protein-derived antioxidant peptides (PIISVYWK, 1004.57Da; FSVVPSPK, 860.09Da) were synthesized by Fmoc SPSS (solid phase peptide synthesis) method (Peptron Inc. Seoul). Other materials used for cell culture were purchased from Gibro-BRL (Gaithersburg, MD, USA). Antibodies for protein analysis (HO-1, Nrf2, β-catenin, PPARγ, SREBP-1, C/EBPa, LPL, FAS, HSL and β-actin) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). and analysis kits used to measure inflammatory cytokines were purchased from GE Healthcare (Buckinghamshire, UK). The assay kits used for the measurement of antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutases) were purchased from Cayman (MI, USA), and other reagents were purchased from Sigma-Aldrich.
세포배양 및 시약 처리Cell culture and reagent processing
실험에 사용된 mouse 골수 유래 중간엽줄기세포(mouse bone marrow-derived mesenchymal stem cell)는 ATCC (D1 cell, CRL-12424)로부터 구입하였고, 10% FBS 및 1% penicillin/streptomycin이 포함된 DMEM 배지를 사용하여 5% CO2 및 37℃ 조건에서 배양하였다. 지방세포분화를 위해서 분화배지(0.5 mM 3-isobutyl-1-methylxanthine, 5 μM dexamethasone, 5 μg/mL insulin 및 10% FBS를 포함하는 DMEM 배지)에서 2일 동안 배양하였다. 2일 동안 배양된 세포는 5 μg/mL insulin, 10% FBS를 포함하는 DMEM 배지에서 추가적 배양하였다. 세포는 실험에 따라 추가적으로 3-21일 동안 배양하였고 배지(5 μg/mL insulin, 10% FBS를 포함하는 DMEM)는 이틀에 한 번씩 교환하면서 펩타이드도 같이 처리하였다.Mouse bone marrow-derived mesenchymal stem cells used in the experiment were purchased from ATCC (D1 cell, CRL-12424), and DMEM medium containing 10% FBS and 1% penicillin/streptomycin was used. 5% CO 2 and incubated at 37 °C conditions. For adipocyte differentiation, it was cultured for 2 days in a differentiation medium (DMEM medium containing 0.5 mM 3-isobutyl-1-methylxanthine, 5 μM dexamethasone, 5 μg/mL insulin, and 10% FBS). Cells cultured for 2 days were additionally cultured in DMEM medium containing 5 μg/mL insulin and 10% FBS. Cells were cultured for an additional 3-21 days according to the experiment, and the medium (DMEM containing 5 μg/mL insulin, 10% FBS) was exchanged every two days, and the peptide was also treated.
HO-1 저해제(5 μM ZnPP)는 2시간 동안 세포에 처리한 후 펩타이드(PIISVYWK 또는 FSVVPSPK)가 포함된 분화배지를 처리하여 지정된 시간 동안 배양하였다.The cells were treated with HO-1 inhibitor (5 μM ZnPP) for 2 hours, and then treated with a differentiation medium containing peptides (PIISVYWK or FSVVPSPK) and cultured for a specified time.
세포독성측정Cytotoxicity measurement
사용된 2종의 펩타이드의 세포독성 측정은 MTT(3-(4,5-dimethythiazol-2-yl)-2,5diphenyltetrazolium bromide)를 이용하였다. 24-well plate에 세포가 70-80% confluent한 상태가 되었을 때 펩타이드를 농도별로 처리한 후 24 또는 48시간 후에 1 mg/mL MTT 용액을 첨가하였다. 4시간 동안 incubator에서 배양한 후 배지를 제거하여 생성된 formazan은 DMSO에 녹여 540nm에서 흡광도를 측정하였다.The cytotoxicity of the two peptides used was measured using MTT (3-(4,5-dimethythiazol-2-yl)-2,5diphenyltetrazolium bromide). When the cells reached a state of 70-80% confluent in a 24-well plate, 1 mg/mL MTT solution was added 24 or 48 hours after each concentration of peptide was treated. After culturing in an incubator for 4 hours, formazan produced by removing the medium was dissolved in DMSO and absorbance was measured at 540 nm.
Oil Red O staining을 이용한 지방 축적 확인Confirmation of fat accumulation using Oil Red O staining
펩타이드(PIISVYWK 및 FSVVPSPK)로 7일 또는 21일 동안 처리 한 후 세포를 PBS로 2회 세척한 후 10% 포르말린으로 1시간 동안 실온에서 고정한 다음 60% 이소프로판올로 세척하였다. 그런 다음 Oil Red O 용액으로 1시간 동안 세포를 염색한 후 증류수로 2회 세척하였다. 중간엽줄기세포에서 지방 축적 이미지는 도립 현미경 (DMI6000, Leica, Wetzlar, Germany)으로 확인하였다. 지방 축적 정량화는 이소프로판올에 지질을 추출한 후 microplate reader(MultiskanTM GO, Thermo ScientificTM, Waltham, MA, USA)를 이용하여 500nm에서 흡광도를 측정하여 계산하였다.After treatment with peptides (PIISVYWK and FSVVPSPK) for 7 or 21 days, the cells were washed twice with PBS, fixed with 10% formalin at room temperature for 1 hour, and then washed with 60% isopropanol. Then, the cells were stained with Oil Red O solution for 1 hour and washed twice with distilled water. Images of fat accumulation in mesenchymal stem cells were confirmed with an inverted microscope (DMI6000, Leica, Wetzlar, Germany). Fat accumulation quantification was calculated by measuring absorbance at 500 nm using a microplate reader (Multiskan TM GO, Thermo Scientific TM , Waltham, MA, USA) after extracting lipids in isopropanol.
지방 분해 측정(lipolysis assay)lipolysis assay
펩타이드(PIISVYWK 및 FSVVPSPK)를 7일 동안 처리한 후, 제조업체의 지침 (Sigma-Aldrich)에 따라 유리 글리세롤 시약(free glycerol reagent)을 사용하여 배양 배지로의 유리 글리세롤 방출을 측정하였다.After the peptides (PIISVYWK and FSVVPSPK) were treated for 7 days, free glycerol release into the culture medium was measured using a free glycerol reagent according to the manufacturer's instructions (Sigma-Aldrich).
세포 내 활성산소종(reactive oxygen species, ROS) 함량 측정Measurement of the content of reactive oxygen species (ROS) in cells
펩타이드(PIISVYWK 및 FSVVPSPK)를 7일 동안 처리 한 후 HBSS 버터에 녹인 20 μM DCFH-DA를 세포에 첨가 한 다음 37℃에서 20분 동안 배양하였다. 세포 내 ROS는 485 및 528 nm의 여기 및 방출에서 형광 강도를 측정하였다.After the peptides (PIISVYWK and FSVVPSPK) were treated for 7 days, 20 μM DCFH-DA dissolved in HBSS butter was added to the cells, and then incubated at 37°C for 20 minutes. Intracellular ROS were measured for fluorescence intensity at excitation and emission at 485 and 528 nm.
항산화 효소 활성 측정Measurement of antioxidant enzyme activity
펩타이드(PIISVYWK 및 FSVVPSPK)를 7일 동안 처리한 후, RIPA buffer(Sigma-Aldrich 社)를 사용하여 세포 용해물을 추출하고 이들 세포 용해물에 포함되어 있는 catalase(CAT), glutathione peroxidase(GPX) 및 superoxide dismutases(SOD)의 활성을 제조사(Cayman, MI, USA)의 지침에 따라 측정하였다.After treatment with the peptides (PIISVYWK and FSVVPSPK) for 7 days, the cell lysate was extracted using RIPA buffer (Sigma-Aldrich), and catalase (CAT), glutathione peroxidase (GPX) and The activity of superoxide dismutases (SOD) was measured according to the manufacturer's instructions (Cayman, MI, USA).
염증성 사이토카인 측정Inflammatory cytokine measurement
펩타이드(PIISVYWK 및 FSVVPSPK)를 7일 동안 처리한 후, 배양 배지에 방출된 염증성 사이토카인(TNF-α, IL-6, IL-1β)는 제조사(GE Healthcare)의 지침에 따라 측정하였다.After treatment with the peptides (PIISVYWK and FSVVPSPK) for 7 days, the inflammatory cytokines (TNF-α, IL-6, IL-1β) released into the culture medium were measured according to the manufacturer's (GE Healthcare) instructions.
면역형광법Immunofluorescence
펩타이드(PIISVYWK 및 FSVVPSPK)를 처리 한 후, 중간엽줄기세포를 4 % 파라포름알데히드로 10분 동안 고정한 다음 0.1 % Triton X-100으로 10 분 동안 투과시켰다. 2% 소혈청 알부민을 사용하여 30분간 blocking 한 후 Nrf2 또는 β-catenin 항체를 사용하여 4℃에서 24시간 동안 배양 한 다음 Alexa Fluor 488-conjugated secondary 항체를 처리하여 1시간 동안 배양하였다. 핵은 10분 동안 2 μg/mL Hoechst 33342로 대조 염색하였고, 형광 신호는 형광 현미경 (Leica)을 사용하여 관찰하였다.After treatment with peptides (PIISVYWK and FSVVPSPK), mesenchymal stem cells were fixed with 4% paraformaldehyde for 10 minutes and then permeabilized with 0.1% Triton X-100 for 10 minutes. After blocking for 30 minutes using 2% bovine serum albumin, Nrf2 or β-catenin antibody was used to incubate for 24 hours at 4°C, and then treated with Alexa Fluor 488-conjugated secondary antibody and incubated for 1 hour. Nuclei were counterstained with 2 μg/mL Hoechst 33342 for 10 min, and the fluorescence signal was observed using a fluorescence microscope (Leica).
Western blottingWestern blotting
펩타이드(PIISVYWK 및 FSVVPSPK)를 7일 동안 처리한 후 RIPA buffer를 이용하여 세포를 파괴한 후 단백질을 추출하여 정량하였다(Pierce BCA assay kit, Thermo Fisher Scientific, MA, USA). 동량의 단백질을 10% SDS-PAGE를 이용하여 분리한 후 PVDF membrane으로 옮겼다. 5% skim milk로 blocking 후 1차 항체를 처리한 후 4℃에서 overnight하였다. 그런 다음 2차 항체를 처리하여 상온에서 2-3시간 반응시킨 후 ECL(enhanced chemiluminescence) assay kit (Pierce Biotechnology, IL, USA)를 이용하여 PVDF membrane 상의 단백질을 검출하였다.Peptides (PIISVYWK and FSVVPSPK) were treated for 7 days, cells were disrupted using RIPA buffer, and proteins were extracted and quantified (Pierce BCA assay kit, Thermo Fisher Scientific, MA, USA). The same amount of protein was separated using 10% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk, the primary antibody was treated and incubated at 4°C overnight. Then, the secondary antibody was treated and reacted at room temperature for 2-3 hours, and then the protein on the PVDF membrane was detected using an enhanced chemiluminescence (ECL) assay kit (Pierce Biotechnology, IL, USA).
<실험결과 및 평가><Experimental results and evaluation>
실시예 1Example 1
지방세포분화 과정에서 홍합단백질 유래 펩타이드가 지방구 형성 및 분해에 미치는 영향Effect of mussel protein-derived peptides on adipocyte formation and degradation during adipocyte differentiation
실시예에 사용된 홍합단백질 유래 펩타이드(PIISVYWK 및 FSVVPSPK)의 중간엽줄기세포의 세포 생존에 미치는 영향을 측정한 결과 펩타이드(10-100μM)에서 어떠한 세포독성도 나타내지 않았다. 홍합단백질 유래 펩타이드가 지방세포분화에 미치는 영향을 조사하기 위해서 중간엽줄기세포가 지방세포로 분화될 때 나타나는 세포내 지방구 형성 정도를 확인하였다.As a result of measuring the effect of the mussel protein-derived peptides (PIISVYWK and FSVVPSPK) on the cell survival of mesenchymal stem cells used in Examples, the peptide (10-100 μM) did not show any cytotoxicity. To investigate the effect of mussel protein-derived peptides on adipocyte differentiation, the degree of intracellular adipocyte formation that occurs when mesenchymal stem cells are differentiated into adipocytes was confirmed.
도 1은 본 발명의 일 실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드가 중간엽줄기세포의 지방세포분화 시 지방 축적 및 지방 분해에 미치는 영향을 도시하고 있다. 도 1의 A는 지방구 형성 현미경 사진이고, 도 1의 B는 지방구 정량, 도 1의 C는 유리 글리세롤 함량을 나타낸다.1 shows the effect of PIISVYWK and FSVVPSPK peptides on fat accumulation and lipolysis during adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention. Fig. 1A is a micrograph of lipid sphere formation, Fig. 1B is the quantification of lipid spheres, and Fig. 1C shows the free glycerol content.
도 1의 A 및 B을 참조하면, 21일 동안 펩타이드가 포함된 분화배지를 처리한 결과 분화배지만 처리한 세포와 비교하여 지방구 형성이 현저히 감소됨을 알 수 있었다. 또한 도 1의 C를 참조하면, 펩타이드 처리에 의한 지방구 감소가 지방 분해로 이어지는지 확인한 결과 펩타이드 처리군에서 지방 분해에 따른 유리 글리세롤의 생성이 증가함을 확인하였다.Referring to FIGS. 1A and 1B , as a result of treatment with a differentiation medium containing peptides for 21 days, it was found that the formation of adipocytes was significantly reduced compared to cells treated with only the differentiation medium. Also, referring to FIG. 1C , as a result of confirming whether the reduction of fat cells by the peptide treatment leads to lipolysis, it was confirmed that the production of free glycerol according to the lipolysis in the peptide-treated group increased.
실시예 2Example 2
지방세포분화 과정에서 홍합단백질 유래 펩타이드가 전사인자 및 지방산 생합성 및 분해 단백질 발현에 미치는 영향Effect of mussel protein-derived peptides on transcription factors and fatty acid biosynthesis and degradation protein expression during adipocyte differentiation
Adipogenesis는 중간엽줄기세포에서 지방전구세포(preadipocyte)가 증식 및 분화되는 과정을 거쳐 성숙한 지방세포(adipocyte)로 되는 과정을 의미하며, 이 과정에서 peroxisome proliferators-activated receptor γ (PPARγ), CCAAT enhancer-binding-proteins α (C/EBPα) 및 sterol regulatory element binding protein-1 (SREBP1) 등의 전사인자 조절의 의해 지방 생성 및 분해에 관여하는 일련의 과정들이 일어난다. 따라서 본 발명에서는 홍합단백질 유래 펩타이드(PIISVYWK 및 FSVVPSPK)가 지방세포분화에 필수적인 전사인자(PPARγ, C/EBPα, SREBP1)에 미치는 영향을 조사하였다.Adipogenesis refers to the process of proliferating and differentiating preadipocytes from mesenchymal stem cells to become mature adipocytes. In this process, peroxisome proliferators-activated receptor γ (PPARγ), CCAAT enhancer- A series of processes involved in adipogenesis and degradation occur by regulation of transcription factors such as binding-proteins α (C/EBPa) and sterol regulatory element binding protein-1 (SREBP1). Therefore, in the present invention, the effect of mussel protein-derived peptides (PIISVYWK and FSVVPSPK) on transcription factors essential for adipocyte differentiation (PPARγ, C/EBPa, SREBP1) was investigated.
도 2는 본 발명의 일 실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드가 중간엽줄기세포의 지방세포분화 시 전사인자 및 지방산 생합성 및 분해 효소들의 발현에 미치는 영향을 도시한 것이다. 도 2의 A는 전사인자 발현, 도 2의 B는 지방산 생합성 및 분해 단백질 발현을 나타낸다.Figure 2 shows the effect of PIISVYWK and FSVVPSPK peptides on the expression of transcription factors and fatty acid biosynthesis and degradation enzymes during adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention. 2A shows transcription factor expression, FIG. 2B shows fatty acid biosynthesis and degradation protein expression.
도 2를 참조하면, 펩타이드를 7일 동안 처리한 결과 세 가지 전사인자의 단백질 발현이 펩타이드 무처리 세포와 비교하여 현저히 줄어드는 것을 확인하였다. 본 발명의 펩타이드 처리에 의한 지방구 형성 감소가 지방산 생합성 및 분해에 영향을 미쳤는지 조사한 결과 펩타이드 처리에 의해 지방산 생합성에 관여하는 효소인 FAS 및 LPL의 발현은 줄어들었고 반면 지질 분해에 관여하는 효소인 HSL의 발현은 증가함을 확인하였다.Referring to FIG. 2 , as a result of treatment with the peptides for 7 days, it was confirmed that the protein expression of three transcription factors was significantly reduced compared to the peptide-untreated cells. As a result of examining whether the reduction in lipocyte formation by the peptide treatment of the present invention had an effect on fatty acid biosynthesis and degradation, the expression of FAS and LPL, enzymes involved in fatty acid biosynthesis, was reduced by the peptide treatment, whereas the enzymes involved in lipid degradation were It was confirmed that the expression of HSL was increased.
실시예 3Example 3
지방세포분화 과정에서 홍합단백질 유래 펩타이드가 염증성 사이토카인 및 활성산소종 생성에 미치는 영향Effect of mussel protein-derived peptides on the production of inflammatory cytokines and reactive oxygen species in the process of adipocyte differentiation
지방세포분화 과정에서 다양한 염증성 사이토카인(TNF-α, IL-6, IL-1β) 및 활성산소종(reactive oxygen species)이 생성되며 이들은 지방세포분화에 중요한 역할을 하는 매개체들이다. In the process of adipocyte differentiation, various inflammatory cytokines (TNF-α, IL-6, IL-1β) and reactive oxygen species are generated, which are mediators that play an important role in adipocyte differentiation.
도 3은 본 발명의 일 실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드 처리가 중간엽줄기세포의 지방세포분화 과정에서 염증성 사이토카인 및 활성산소종 생성에 미치는 영향을 도시한 것이다. 도 3의 A~C는 염증성 사이토카인 함량, 도 3의 D~E는 세포내 활성산소종(현미경 사진 및 정량)을 나타낸다. 도 3을 참조하면, 펩타이드 처리 후 이들 염증성 사이토카인 및 활성산소종 생성을 측정한 결과 펩타이드 처리가 지방세포분화 과정에서 생성되는 염증성 사이토카인 및 활성산소종을 효과적을 억제함을 알 수 있다.Figure 3 shows the effect of PIISVYWK and FSVVPSPK peptide treatment on the production of inflammatory cytokines and reactive oxygen species in the process of adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention. 3A to 3C show the content of inflammatory cytokines, and FIG. 3D to E show intracellular reactive oxygen species (micrographs and quantitative). Referring to FIG. 3 , as a result of measuring the production of these inflammatory cytokines and reactive oxygen species after the peptide treatment, it can be seen that the peptide treatment effectively inhibits the inflammatory cytokines and reactive oxygen species produced during adipocyte differentiation.
실시예 4Example 4
지방세포분화 과정에서 홍합단백질 유래 펩타이드 처리가 HO-1 발현 및 전사인자 활성화에 미치는 영향Effect of mussel protein-derived peptide treatment on HO-1 expression and transcription factor activation during adipocyte differentiation
본 발명의 펩타이드 처리가 지방세포분화 과정에서 HO-1의 발현에 미치는 영향을 조사하였다. 도 4는 본 발명의 일 실시예에 따라 PIISVYWK 및 FSVVPSPK 펩타이드가 중간엽줄기세포의 지방세포분화 과정에서 HO-1 발현 및 전사인자 활성화에 미치는 영향을 도시한 것이다. 도 4의 A는 HO-1 발현, 도 4의 B는 전사인자 Nrf2 활성화를 나타낸다.The effect of the peptide treatment of the present invention on the expression of HO-1 in adipocyte differentiation was investigated. Figure 4 shows the effect of PIISVYWK and FSVVPSPK peptides on HO-1 expression and transcription factor activation in the process of adipocyte differentiation of mesenchymal stem cells according to an embodiment of the present invention. 4A shows HO-1 expression, and FIG. 4B shows activation of the transcription factor Nrf2.
도 4를 참조하면, 펩타이드 처리에 의해서 HO-1 단백질 발현이 무처리군(control)과 비교하여 현저히 증가하였고 HO-1 발현에 필요한 전사인자(Nrf2)의 활성화(핵이동)가 일어나는 것을 확인하였다. 이러한 결과는 펩타이드 처리에 의한 HO-1의 발현이 지방세포분화 과정에서 발생하는 염증성 사이토카인 및 활성산소종의 생성을 억제하는 것으로 판단된다. 4, it was confirmed that HO-1 protein expression was significantly increased by peptide treatment compared to the untreated group (control), and activation (nuclear migration) of the transcription factor (Nrf2) required for HO-1 expression occurred. . These results indicate that the expression of HO-1 by peptide treatment suppresses the production of inflammatory cytokines and reactive oxygen species that occur during adipocyte differentiation.
실시예 5Example 5
홍합단백질 유래 펩타이드에 의한 HO-1 발현이 지방세포분화 과정에 미치는 영향Effect of HO-1 expression by mussel protein-derived peptide on adipocyte differentiation process
펩타이드 처리에 의한 HO-1 발현이 지방세포분화를 조절한다는 것을 증명하기 위하여 HO-1 저해제(ZnPP, 5 μM)를 사용하여 지방세포분화에 관여하는 지방구 형성, 전사인자, 염증성 사이토카인, 활성산소종 생성에 미치는 영향을 조사하였다. To prove that HO-1 expression by peptide treatment regulates adipocyte differentiation, adipocyte formation, transcription factor, inflammatory cytokine, and activity involved in adipocyte differentiation using HO-1 inhibitor (ZnPP, 5 μM) The effect on oxygen species production was investigated.
도 5는 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 지방구 형성 및 분해에 미치는 영향을 도시한 것이다. 도 5의 A는 지방구 형성 현미경 사진, 도 5의 B는 지방구 정량, 도 5의 C는 유리 글리세롤 정량을 나타낸다. 5 shows the effect of HO-1 inhibitor treatment on adipocyte formation and degradation during adipocyte differentiation according to an embodiment of the present invention. Fig. 5A is a micrograph of lipid sphere formation, Fig. 5B is a quantitation of fat spheres, and Fig. 5C shows quantification of free glycerol.
도 5를 참조하면, 먼저 HO-1 저해제 처리 시 지방구 형성에 미치는 영향을 조사한 결과 펩타이드 처리에 따라 줄어든 지방구 형성이 HO-1 저해제를 처리한 결과 지방구 형성이 증가함을 알 수 있었다. 또한 줄어든 지방구가 지방 분해로 이어지는지를 유리 글리세롤 측정으로 확인한 결과 펩타이드 처리에 의해서 증가한 유리 글리세롤 함량이 HO-1 저해제 처리에 의하여 줄어드는 것을 확인할 수 있었다.Referring to FIG. 5 , first, as a result of examining the effect of HO-1 inhibitor treatment on adipocyte formation, it was found that the reduction in adipocyte formation according to the peptide treatment increased the adipocyte formation as a result of treatment with the HO-1 inhibitor. In addition, as a result of confirming whether the reduced fat cells lead to lipolysis by free glycerol measurement, it was confirmed that the free glycerol content increased by the peptide treatment was reduced by the HO-1 inhibitor treatment.
본 실시예에서는 펩타이드 처리에 의한 HO-1 발현이 β-catenin의 활성화(핵이동) 및 HO-1 저해제 처리에 의한 β-catenin의 활성화에 미치는 영향을 조사하였다. In this example, the effect of HO-1 expression by peptide treatment on activation of β-catenin (nuclear migration) and β-catenin activation by HO-1 inhibitor treatment was investigated.
도 6은 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 β-catenin 활성화에 미치는 영향을 도시한 것이다. 도 6을 참조하면, 펩타이드 처리에 의해서 β-catenin 전사인자의 핵이동을 증가시키는 것으로 나타났으나 HO-1 저해제를 처리한 결과 펩타이드 처리에 의한 β-catenin 전사인자의 핵이동이 억제됨을 알 수 있었다. 즉 펩타이드 처리에 따른 HO-1 발현이 β-catenin 전사인자 활성화에 관여한다는 것을 알 수 있다.6 shows the effect of HO-1 inhibitor treatment on β-catenin activation in the process of adipocyte differentiation according to an embodiment of the present invention. Referring to FIG. 6 , it was shown that the nuclear migration of the β-catenin transcription factor was increased by the peptide treatment, but as a result of the treatment with the HO-1 inhibitor, it can be seen that the nuclear migration of the β-catenin transcription factor by the peptide treatment was suppressed. there was. That is, it can be seen that the expression of HO-1 according to the peptide treatment is involved in the activation of the β-catenin transcription factor.
도 7은 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 활성산소종 및 염증성 사이토카인 생성에 미치는 영향을 도시한 것이다. 도 7을 참조하면, HO-1 저해제 처리가 활성산소종 및 염증성 사이토카인 생성에 미치는 영향을 조한 결과 펩타이드 처리에 의해서 억제되었던 활성산소종 및 염증성 사이토카인 생성이 다시 증가됨을 알 수 있었다. 이는 펩타이드 처리에 의한 HO-1 발현이 지방세포분화 조절에 있어 중요한 역할을 한다는 것을 확인할 수 있다.7 shows the effect of HO-1 inhibitor treatment on the production of reactive oxygen species and inflammatory cytokines in the process of adipocyte differentiation according to an embodiment of the present invention. Referring to FIG. 7 , as a result of examining the effect of HO-1 inhibitor treatment on reactive oxygen species and inflammatory cytokine production, it was found that reactive oxygen species and inflammatory cytokine production, which were inhibited by peptide treatment, were increased again. This confirms that HO-1 expression by peptide treatment plays an important role in regulating adipocyte differentiation.
시예 6Example 6
홍합단백질 유래 펩타이드에 의한 HO-1 발현이 지방세포분화에 관여하는 전사인자 발현에 미치는 영향Effect of HO-1 expression by mussel protein-derived peptide on the expression of transcription factors involved in adipocyte differentiation
이상과 같이 펩타이드 처리에 의한 HO-1 발현이 지방세포분화에 있어서 지방구 형성, 활성산소종, 염증성 사이토카인 등을 조절하는 것으로 확인되었다. 또한 HO-1 저해제 처리가 HO-1 발현 및 그에 따른 지방세포분화에 필수적인 전사인자 발현에 미치는 영향을 확인하였다. As described above, it was confirmed that HO-1 expression by peptide treatment regulates adipocyte formation, reactive oxygen species, and inflammatory cytokines in adipocyte differentiation. In addition, the effect of HO-1 inhibitor treatment on HO-1 expression and thus on the expression of transcription factors essential for adipocyte differentiation was confirmed.
도 8은 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 HO-1 및 전사인자 단백질 발현에 미치는 영향을 도시한 것이다. 도 8의 A를 참조하면, 펩타이드 처리에 의해서 증가되었던 HO-1의 발현이 저해제를 처리한 결과 HO-1 발현이 감소됨을 알 수 있었다. 또한 도 8의 B를 참조하면, HO-1 저해제 처리가 전사인자 발현에 미치는 영향을 확인한 결과 펩타이드 처리에 의해 감소된 세 가지 전사인자의 발현이 HO-1 저해제 처리에 의해서 증가됨을 확인할 수 있었다. 8 shows the effect of HO-1 inhibitor treatment on HO-1 and transcription factor protein expression during adipocyte differentiation according to an embodiment of the present invention. Referring to FIG. 8A , it was found that the expression of HO-1 increased by peptide treatment was decreased as a result of treatment with the inhibitor. Also, referring to FIG. 8B , as a result of confirming the effect of HO-1 inhibitor treatment on transcription factor expression, it was confirmed that the expression of three transcription factors decreased by peptide treatment was increased by HO-1 inhibitor treatment.
실시예 7Example 7
홍합단백질 유래 펩타이드에 의한 HO-1 발현이 p-AMPK/AMPK 발현에 미치는 영향Effect of HO-1 expression by mussel protein-derived peptide on p-AMPK/AMPK expression
비만은 에너지 불균형과 관련된 대사 장애이다. AMPK(AMP-activated protein kinase)는 최근 항비만 물질 개발을 위한 신규 타켓으로 주목받고 있는데 세포 내 에너지 저장 및 소비와 관련된 중요한 에너지 센서로서 작용한다. 활성화된 AMPK는 몇몇 하위 단계에 있는 기질을 인산화하여 지방산 합성과 콜레스테롤 합성을 억제한다. AMPK의 활성을 증가시키는 물질은 ATP 합성촉진, 글루코스 흡수의 증가, 지방의 β-산화(oxidation) 촉진, 콜레스테롤 합성 저해, 염증 반응 완화 등의 효과가 있는 것으로 보고되고 있어 비만을 포함한 당뇨, 인슐린 저항성, 고지혈증, 염증 등 대사성 질환 치료제 개발을 위한 약물 표적으로 활용되고 있다. 또한 최근 연구들에 따르면, AMPK의 활성화가 지방세포분화에 관여하는 전자인자(PPARγ, SREBP-1, C/EBPα)의 발현을 억제하여 지방세포분화를 억제하는 것으로 나타났다. 또한 HO-1 발현의 항비만 효과를 증명하기 위하여 HO-1 저해제 처리가 AMPK 활성화에 미치는 영향을 확인하였다. Obesity is a metabolic disorder associated with energy imbalance. AMPK (AMP-activated protein kinase) has recently been attracting attention as a new target for the development of anti-obesity substances, and acts as an important energy sensor related to energy storage and consumption in cells. Activated AMPK inhibits fatty acid synthesis and cholesterol synthesis by phosphorylating substrates at several lower levels. Substances that increase the activity of AMPK have been reported to have effects such as promotion of ATP synthesis, increase of glucose absorption, promotion of β-oxidation of fat, inhibition of cholesterol synthesis, and alleviation of inflammatory response. It is being used as a drug target for the development of therapeutics for metabolic diseases such as , hyperlipidemia, and inflammation. Also, recent studies have shown that activation of AMPK inhibits adipocyte differentiation by suppressing the expression of electron factors (PPARγ, SREBP-1, C/EBPa) involved in adipocyte differentiation. In addition, in order to prove the anti-obesity effect of HO-1 expression, the effect of HO-1 inhibitor treatment on AMPK activation was confirmed.
도 9는 본 발명의 일 실시예에 따라 지방세포분화 과정에서 HO-1 저해제 처리가 AMPK 활성화에 미치는 영향을 도시한 것이다. 도 9를 참조하면, 펩타이드 처리가 지방세포분화 과정에서 AMPK의 인산화(p-AMPK)를 증가시키는 것으로 나타났으며, 이는 HO-1의 저해제를 처리할 시 AMPK의 인산화 발현이 감소됨을 확인하였다. 이는 펩타이드 처리에 의한 HO-1의 발현이 지방세포분화 조절 및 지방 분해를 통한 지방 축적을 억제한다는 것으로 볼 수 있다.9 shows the effect of HO-1 inhibitor treatment on AMPK activation during adipocyte differentiation according to an embodiment of the present invention. Referring to FIG. 9 , it was found that the peptide treatment increased AMPK phosphorylation (p-AMPK) in the process of adipocyte differentiation, which confirmed that the phosphorylation expression of AMPK was reduced when the HO-1 inhibitor was treated. It can be seen that the expression of HO-1 by peptide treatment inhibits fat accumulation through adipocyte differentiation and lipolysis.
이상의 결과를 종합하면 홍합단백질 유래 펩타이드(PIISVYWK 및 FSVVPSPK)가 지방세포분화를 억제함을 확인하였는데 이는 지방세포분화 과정에서 펩타이드 처리에 의한 HO-1의 발현이 Wnt/β-catenin의 활성화를 유도하며 이는 지방세포분화에 필수적인 전사인자를 조절하여 지방세포분화를 억제하며 또한 p-AMPK 활성화를 통하여 지방 축적을 억제하는 것으로 나타났다.Summarizing the above results, it was confirmed that mussel protein-derived peptides (PIISVYWK and FSVVPSPK) inhibited adipocyte differentiation. It was shown to inhibit adipocyte differentiation by regulating transcription factors essential for adipocyte differentiation, and also inhibit fat accumulation through p-AMPK activation.
실시예의 결과로부터 본 발명은 홍합단백질 유래 펩타이드가 지방세포분화에 관여하는 전자인자인 PPARγ 및 C/EBPα의 발현을 억제하며 지방세포에서 지방분해를 촉진하여 세포내 지방의 과잉 축적을 억제함을 확인하였다. 또는 지방산 생합성에 관여하는 단백질인 SREBP-1, LPL, FAS 등을 억제함과 동시에 지방 분해를 촉진하는 p-AMPK를 활성화 시키는 것을 확인하였다. From the results of the Examples, it was confirmed that the mussel protein-derived peptide suppresses the expression of PPARγ and C/EBPa, which are electronic factors involved in adipocyte differentiation, and promotes lipolysis in adipocytes, thereby suppressing excessive accumulation of intracellular fat. did. Alternatively, it was confirmed that it inhibits SREBP-1, LPL, FAS, etc., which are proteins involved in fatty acid biosynthesis, and simultaneously activates p-AMPK, which promotes lipolysis.
또한 본 발명에서는 새로운 분자 target으로 HO-1의 역할을 확인한 결과 홍합단백질 유래 펩타이드가 지방세포분화 과정에서 HO-1의 발현을 증가시켜 앞서 언급한 전사인자 및 단백질 발현을 억제함을 확인하였다. In addition, in the present invention, as a result of confirming the role of HO-1 as a new molecular target, it was confirmed that the mussel protein-derived peptide increased the expression of HO-1 during adipocyte differentiation, thereby inhibiting the expression of the aforementioned transcription factors and proteins.
Claims (7)
상기 펩타이드는 홍합단백질로부터 유래하는 것을 특징으로 하는 지방세포분화 억제용 조성물.The method of claim 1,
The composition for inhibiting adipocyte differentiation, characterized in that the peptide is derived from mussel protein.
상기 펩타이드에 의하여 heme oxygenase-1(HO-1)의 발현을 증가시키고 β-catenin의 활성화를 유도하여 지방세포분화를 억제하는 것을 특징으로 하는 지방세포분화 억제용 조성물.The method of claim 1,
A composition for inhibiting adipocyte differentiation, characterized in that the peptide inhibits adipocyte differentiation by increasing the expression of heme oxygenase-1 (HO-1) and inducing the activation of β-catenin.
상기 펩타이드는 전사인자인 PPARγ 및 C/EBPα를 억제하여 지방세포분화를 조절하는 것을 특징으로 하는 지방세포분화 억제용 조성물.The method of claim 1,
The composition for inhibiting adipocyte differentiation, characterized in that the peptide regulates adipocyte differentiation by inhibiting transcription factors PPARγ and C/EBPa.
상기 펩타이드는 p-AMPK 활성화에 의하여 지방분해를 촉진하는 것을 특징으로 하는 지방분해 촉진용 조성물.
7. The method of claim 6,
The composition for promoting lipolysis, characterized in that the peptide promotes lipolysis by activation of p-AMPK.
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