KR101510257B1 - Diabetic renal fibrosis or tubulointersitial fibrosis inhibiting composition of chrysin - Google Patents
Diabetic renal fibrosis or tubulointersitial fibrosis inhibiting composition of chrysin Download PDFInfo
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본 발명은 크리신을 유효성분으로 함유하는 신세뇨관 섬유화로 인한 신장섬유증 억제용 조성물 대한 것으로, 더 상세하게는 당뇨합병증으로 초래되는 신세뇨관 섬유화로 인한 신장섬유증 억제용 조성물에 직접적으로 관련된 인간 신장 근위세뇨관 상피세포 (Human Renal Proximal Tubular Epithelial cell, RPTEC)를 고혈당에 노출시키고 여기에 크리신을 처리하여 배양함으로써 신세뇨관 상피세포가 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT)으로 인하여 결합조직 성장인자(Connetive Tissue Growth Factor, CTGF)와, 제 4형 콜라겐(Collagen)의 생성을 감소시키고, EMT 관련 단백질 바이오마커인 N-cadherin의 발현을 억제시키며, EMT를 억제하는 세포부착단백질인 E-cadherin의 발현을 증가시켜 만성 고혈당으로 인하여 신세뇨관 섬유화로 인한 신장섬유증을 개선시킬 수 있는 크리신을 유효성분으로 함유하는 조성물에 대한 것이다.The present invention relates to a composition for inhibiting renal fibrosis caused by renin tubular fibrosis comprising chrysin as an active ingredient, and more particularly, to a composition for inhibiting renal fibrosis caused by renal tubular fibrosis caused by diabetic complications, Epithelial-Mesenchymal Transition (EMT) has been implicated in the development of renal tubular epithelial cells (HSCs) by exposing epidermal cells (RPEECs) to hyperglycemia, Cadherin, a cell attachment protein that inhibits EMT-related protein biomarker N-cadherin expression and reduces the production of collagen and type 4 collagen (Connetive Tissue Growth Factor, CTGF) Which can improve renal fibrosis due to renal tubular fibrosis due to increased hyperglycemia The present invention relates to a composition containing lysine as an active ingredient.
당뇨병은 인슐린의 분비량이 부족하거나 정상적인 기능이 이루어지지 않는 등의 대사질환의 일종으로, 혈중 포도당의 농도가 높아지는 고혈당을 특징으로 하며, 고혈당으로 인하여 여러 증상 및 징후를 일으키고 소변에서 포도당을 배출하게 된다. Diabetes mellitus is a type of metabolic disease that lacks insulin secretion or does not function normally. It is characterized by hyperglycemia in which the concentration of glucose in the blood rises. Hyperglycemia causes various symptoms and signs and excretes glucose from the urine .
또한, 당뇨병 환자가 장기간 고혈당에 노출되었을 경우에는 말초신경증, 자율신경증, 대 혈관 합병증, 미세혈관 합병증을 비롯하여 기타 만성합병증을 초래할 수 있다. 미세혈관 합병증의 하나인 당뇨병성 신증의 초기변화는 신장의 용적이 증가하고 단백뇨 배설이 증가하며, 사구체 및 세뇨관 기저막의 비후, 이후 사구체 혈관간질 증가, 세포 외 지질의 축척, 사구체 경화증 및 세뇨관 간질의 섬유화로 진행하게 된다. In addition, when diabetic patients are exposed to prolonged hyperglycemia, they may cause peripheral neuropathy, autonomic neuropathy, macrovascular complications, microvascular complications, and other chronic complications. Early changes in diabetic nephropathy, one of the microvascular complications, include increased renal volume, increased proteinuria excretion, increased glomerular and tubular basement membrane thickening, increased glomerular vascular epilepsy, extracellular lipid accumulation, glomerular sclerosis and tubular epilepsy And proceeds to fibrosis.
신장의 세뇨관 및 간질은 전체 신장 용적의 80%를 차지하며 이중 간질이 차지하는 부분은 10-17%에 이른다고 알려져 있다. 간질은 세뇨관, 혈관, 사구체, mesangium과 밀접하게 연결되어 있어서 이들 주변 지역의 병적인 문제가 쉽게 간질에 영향을 미치게 되고, 아울러 세뇨관 간질의 병적인 변화가 사구체의 병변보다 신장 기능의 예후와 밀접한 연관이 있다고 알려져 있다. 일반적으로 renal fibrogenesis의 과정은 유도(induction), 기질 생성(matrix synthesis), 및 분해(resolution)로 나뉘게 되는데 섬유화는 섬유아세포(fibroblast)의 이동과 증식으로 유도되며, collagen, fibronectin, 여러 proteoglycan과 같은 간질성 기질 단백(interstitialmatrix protein)의 생성과 축적으로 일어나게 된다. 세포외기질(extracellular matrix, ECM)의 축적은 정상적인 상처 치유 과정의 일부인 반면 섬유화는 만성적이고 심한 염증에 대한 병적인 반응이다. 신장이 염증 또는 손상을 받게 되면, 신장에 들어오거나 신장내의 상주하는 세포에서 유리되는 여러 인자들이 신장 조직에서 세포외 기질의 생산을 자극하게 되고, 결국 이러한 세포외 기질 물질의 과다 생성은 세포의 경화를 유발하고 신장의 구조 또는 기능상의 손상을 가져 오게 된다. The renal tubules and epilepsy account for 80% of the total renal volume, and epilepsy accounts for 10-17%. Because epilepsy is closely related to the tubules, blood vessels, glomeruli, and mesangium, pathological problems in these peripheral areas can easily affect epilepsy, and pathological changes of tubular epilepsy are more closely related to the prognosis of renal function . In general, the process of renal fibrogenesis is divided into induction, matrix synthesis, and resolution. Fibrosis is induced by the migration and proliferation of fibroblasts, and is induced by collagen, fibronectin, several proteoglycan It is caused by the production and accumulation of interstitial matrix proteins. The accumulation of extracellular matrix (ECM) is part of the normal wound healing process, whereas fibrosis is a pathological response to chronic and severe inflammation. When kidneys become inflamed or damaged, many factors that enter the kidneys or are released from the resident cells in the kidneys stimulate the production of extracellular matrix in kidney tissue, and ultimately, And causes damage to the structure or function of the kidneys.
지속적인 고혈당 자극으로 인한 신세뇨관 상피세포의 EMT 과정에서는 완전히 분화된 상피세포가 섬유아세포로 형질적인 변이(phenotypic change)가 일어나게 되는데, 이 과정에서는 원래 상피세포의 특징인 극성이 소실되면서 다변형의 세포모양이 중배엽세포의 모양인 길쭉하게 늘어난 모양으로 바뀌며 상피세포간의 접착(tight junction)과 결합(adherens junction)이 상실되며 운동성과 수축성이 증가하게 된다. 이 과정에서 세포내에서 발현되는 단백질의 표현형에도 변화가 생기게 되는데, 세포의 adherens junction을 주도하는 E-cadherin 단백질 발현이 감소하게 되고, 중배엽 세포의 접착 단백질인 N-cadherin 단백질의 발현이 증가하게 된다. 뿐만 아니라 세포와 세포외기질을 이루고 있는 기질 단백질인 4형 콜라겐(collagen)과 결합조직 성장인자(Connective Tissue Growth factor, CTGF)의 발현이 크게 증가하게 되는데 이러한 단백질 발현의 변화와, 기질단백질의 분비는 EMT에 의한 신세뇨관 간질 섬유화에 중요한 역할을 하는 것으로 알려져 있다.In the EMT process of renal tubular epithelial cells due to persistent hyperglycemic stimulation, a phenotypic change occurs in the completely differentiated epithelium cells. In this process, the characteristic polarity of the epithelial cells is lost, The shape changes into an elongated shape of mesenchymal cells, and the tight junctions and adherence junctions between epithelial cells are lost, resulting in increased mobility and contractility. In this process, the phenotype of the protein expressed in the cell is also changed. The expression of E-cadherin protein, which leads to the adherence junction of the cell, is decreased, and the expression of N-cadherin protein, an adhesive protein of mesodermal cells, is increased . In addition, expression of collagen type 4 and connective tissue growth factor (CTGF), which are matrix proteins that constitute the extracellular matrix of cells, are greatly increased. Such changes in protein expression and secretion of substrate protein Is known to play an important role in the development of renal tubular interstitial fibrosis by EMT.
크리신은 식물에 광범위하게 분포되어 있는 flavonoid의 일종으로 항산화, 항염증, 항암, 항 알레르기 작용 등 다양한 생리활성이 보고되어 있는 생리활성 물질이다. 현재는 근육강화제로 시판되어 널리 쓰이고 있는 성분 중의 하나이며, 최근에는 항 불안효과, 항 경련효능에 대한 연구가 알려져 있으며, 크리신 유도체들을 이용한 항 당뇨 활성에 대한 연구가 진행 중에 있다.Chrysin is a flavonoid that is widely distributed in plants. It is a physiologically active substance that has been reported to have various physiological activities such as antioxidant, anti-inflammation, anti-cancer and antiallergic action. Currently, it is one of the widely used ingredients that are commercially available as muscle strengthening agents. Recently, studies on anti-anxiety and anti-convulsive effects have been known, and studies on anti-diabetic activity using chrysin derivatives are underway.
현재까지 혈관 및 신장합병증과 관련된 당뇨 합병증의 예방 및 치료를 위한 연구가 활발하게 이루어지고 있으며, 장기간 고혈당 노출에 의한 신세뇨관 섬유화로 인한 신장경화증 완화를 위한 다양한 연구가 진행 중에 있다. 그러나 식이 성분을 포함한 천연 성분을 이용하여 신장섬유증과 신세뇨관 경화증을 예방하고 치료하는 연구는 미흡한 실정이다. 따라서 결합조직 성장인자(CTGF)의 활성을 억제시키고 제 4형 콜라겐 생성을 감소시키며 EMT 관련 단백질 바이오마커인 N-cadherin의 발현 감소 및 EMT를 억제하는 세포부착 단백질인 E-cadherin의 발현을 증가시켜 만성 고혈당으로 인한 신세뇨관 섬유화로 인한 신장 섬유증을 개선시킬 수 있는 천연성분에 대한 연구가 필요하다.To date, various studies have been conducted for the prevention and treatment of diabetic complications related to vascular and renal complications, and various studies are under way to alleviate renal sclerosis due to renal tubular fibrosis due to prolonged exposure to hyperglycemia. However, studies on the prevention and treatment of renal fibrosis and renal tubular sclerosis using natural ingredients including dietary ingredients are insufficient. Therefore, it inhibits the activity of connective tissue growth factor (CTGF), decreases the formation of type-IV collagen, increases the expression of N-cadherin, an EMT-related protein biomarker, and the expression of E-cadherin, There is a need for studies on natural ingredients that can improve renal fibrosis due to renal tubular fibrosis due to chronic hyperglycemia.
본 발명은 상기와 같은 문제를 해결하고자 발명한 것으로,The present invention has been made to solve the above-mentioned problems,
그 목적은 당뇨 합병증으로 인하여 초래되는 신세뇨관 섬유화로 인한 신장경화증의 주요원인인 고혈당에 노출시킨 인간 신장 근위세뇨관 상피세포 (Human Renal Proximal Tubular Epithelial cell, RPTEC)의 결합조직 성장인자(CTGF)의 활성 억제, 제 4형 콜라겐(Collagen) 생성 감소, 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT) 관련 단백질 marker인 N-cadherin의 발현 감소 및 상피-중간엽세포로의 이행을 억제하는 세포부착 단백질인 E-cadherin의 발현을 증가시킴으로써 만성 고혈당으로 인한 신장섬유증과 신세뇨관 경화증을 개선시킬 수 있는 크리신을 함유한 조성물을 제공하는 데 있다.The purpose of this study was to investigate the effect of CTGF on human renal proximal tubular epithelial cells (RPTEC) exposed to hyperglycemia, which is the main cause of renal sclerosis caused by diabetic complications. Cell adhesion that inhibits the expression of N-cadherin, which is a protein marker related to epithelial-mesenchymal transition (EMT) and epithelial-mesenchymal cells, and inhibits collagen formation by type 4 collagen, Which is capable of improving renal fibrosis and renoscopic sclerosis due to chronic hyperglycemia by increasing the expression of E-cadherin, a protein of the present invention.
상기와 같은 목적을 달성하기 위하여 신장섬유증과 신세뇨관 경화증에 직접적으로 관련된 인간 신장 근위세뇨관 상피세포 (Human Renal Proximal Tubular Epithelial cell, RPTEC)를 고혈당에 노출시키고 여기에 크리신을 농도별(1-20M)로 처리하여 함께 배양함으로써, 결합조직 성장인자(CTGF)의 활성 억제, 제 4형 콜라겐(Collagen) 생성 감소, 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT) 관련 단백질 바이오 마커인 N-cadherin의 발현 감소 및 EMT를 억제하는 세포부착 단백질인 E-cadherin의 발현을 증가시킴으로써 만성 고혈당의 결과로 나타나는 신세뇨관 섬유화에 의한 신장 섬유증을 개선시킬 수 있다.In order to achieve the above object, human renal proximal tubular epithelial cells (RPTEC) directly associated with renal fibrosis and renal tubular sclerosis were exposed to hyperglycemia, (N-cadherin), which is a protein biomarker related to epithelial-mesenchymal transition (EMT) -related protein, inhibits the activity of CTGF, inhibits formation of collagen type 4, And the expression of E-cadherin, a cell attachment protein that inhibits EMT, thereby improving renal fibrosis caused by renal tubular fibrosis, which is a result of chronic hyperglycemia.
본 발명에 따른 크리신을 함유한 조성물을 이용함으로써, 결합조직 성장인자(CTGF)의 활성 억제, 제 4형 콜라겐(Collagen) 생성 감소, 상피-중간엽세포 이행으로 인한 접착단백질인 E-cadherin의 증가, EMT 관련 단백질인 N-cadherin의 발현을 감소시켜 당뇨합병증으로 초래되는 신세뇨관 섬유화로 인한 신장 섬유증을 개선시키는 효과가 있다.By using the composition containing the chrysin according to the present invention, it is possible to inhibit the activity of connective tissue growth factor (CTGF), decrease the formation of collagen type 4, increase the adhesion protein E-cadherin due to epithelial-mesenchymal cell migration , And the expression of N-cadherin, an EMT-related protein, is reduced, thereby improving renal fibrosis caused by renal tubular fibrosis caused by diabetic complications.
도 1은 크리신을 인간 신장 근위세뇨관 상피세포 (RPTEC)에 처리하여 배양하였을 때, 만성 고혈당에서 결합조직 성장인자(CTGF)의 발현 및 분비 억제 활성을 나타낸 웨스턴 블롯 분석결과이며, 이것을 덴시토미터(densitometer)로 측정하여 정량화한 것이다.
도 2는 크리신을 인간 신장 근위세뇨관 상피세포 (RPTEC)에 처리하여 배양하였을 때, 만성 고혈당에서 제 4형 콜라겐 생성에 대한 억제활성을 나타낸 웨스턴 블롯 분석결과이며, 이것을 덴시토미터로 측정하여 정량화한 것이다.
도 3은 크리신을 인간 신장 근위세뇨관 상피세포 (RPTEC)에 처리하여 배양하였을 때, 만성 고혈당에서 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT) 관련 단백질 바이오 마커인 N-cadherin의 발현 감소 및 EMT로의 이행을 억제하는 세포부착 단백질인 E-cadherin의 발현을 증가시키는 활성을 나타낸 웨스턴 블롯 분석결과이며, 이것을 덴시토미터로 측정하여 정량화한 것이다.FIG. 1 shows the result of Western blot analysis showing the expression and secretion inhibitory activity of connective tissue growth factor (CTGF) in chronic hyperglycemia when cultured by treating the human kidney proximal tubular epithelial cells (RPTEC) with chrysin, densitometer).
FIG. 2 is a Western blot analysis showing the inhibitory activity against formation of type-4 collagen in chronic hyperglycemia when cultured by treating the human kidney proximal tubular epithelial cells (RPTEC) with chrysin. The result is quantified by measuring with a densitometer will be.
FIG. 3 shows the expression of N-cadherin, an Epithelial-Mesenchymal Transition (EMT) -related protein biomarker in chronic hyperglycemia, when cultured with human kidney proximal tubular epithelial cells (RPTEC) Western blot analysis showing the activity of increasing the expression of E-cadherin, a cell adhesion protein inhibiting the transition to EMT, was quantified by measuring with a densitometer.
본 발명은 당뇨합병증으로 인해 초래되는 신세뇨관 섬유화에 의한 신장섬유증 억제 활성을 갖는 크리신을 유효성분으로 함유하는 조성물에 대한 것이다.
The present invention relates to a composition containing, as an active ingredient, chrysin having an activity of inhibiting renal fibrosis caused by renal tubular fibrosis caused by diabetic complications.
더 상세하게는 당뇨합병증으로 초래되는 신장섬유증에 직접적으로 관련된 인간 신장 근위세뇨관 상피세포 (Human Renal Proximal Tubular Epithelial cell, RPTEC)를 고혈당에 노출시키고 여기에 크리신을 처리하여 배양함으로써 신세뇨관 상피세포의 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT)으로 인한 세포외기질에서의 결합조직 성장인자(Connetive Tissue Growth Factor, CTGF)와 제 4형 콜라겐(Collagen) 생성을 감소시키고, EMT로 인한 접착단백질인 E-cadherin를 증가시키며, EMT 관련 단백질인 N-cadherin의 발현을 감소시켜 만성 고혈당으로 인한 신세뇨관 섬유화로 인한 신장 섬유증을 개선시킬 수 있는 크리신을 유효성분으로 함유하는 조성물에 대한 것이다.
More specifically, human renal proximal tubular epithelial cells (RPTEC), which are directly related to renal fibrosis caused by diabetic complications, are exposed to hyperglycemia and cultured by treatment with chrysin, whereby the epithelium of the renal tubular epithelium - Reduction of Connnet Tissue Growth Factor (CTGF) and type 4 collagen production in extracellular matrix due to Epithelial-Mesenchymal Transition (EMT) The present invention relates to a composition containing chrysin as an active ingredient capable of increasing E-cadherin and reducing renal fibrosis caused by renal tubular fibrosis caused by chronic hyperglycemia by reducing the expression of EMT-related protein N-cadherin.
크리신은 하기 화학식 1과 같은 구조를 가지고 있고 통상적인 방법에 따라 제조하여 사용하거나 시판되는 것을 구입하여 사용할 수 있다.
The chrysene has a structure as shown in the following Chemical Formula 1, and can be prepared according to a conventional method or commercially available.
화학식 IFormula I
크리신(5,7-Dihydroxy-2-phenyl-4H-chromen-4-one)
(5,7-Dihydroxy-2-phenyl-4H-chromen-4-one)
본 발명에 의한 당뇨합병증으로 인해 초래되는 신세뇨관 섬유화로 인한 신장섬유증에 억제 활성을 갖는 크리신(chrysin)을 유효성분으로 함유한 조성물은 그 제형에 있어서 특별히 한정되지 않지만, 바람직하게는 음료, 분말, 농축, 캡슐, 의약으로 하는 것이다.
The composition containing chrysin having an inhibitory activity on renal fibrosis caused by renal tubular fibrosis caused by diabetic complication according to the present invention as an active ingredient is not particularly limited in its formulation, , Concentrates, capsules, and medicines.
이하에서 실시예를 통하여 본 발명을 보다 구체적으로 설명한다. 그러나 하기의 실시예는 본 발명을 구체적으로 예시하기 위한 것일 뿐, 본 발명의 권리범위를 제한하는 것이 아님은 명백한 사실이다. 즉, 본 발명의 단순한 변형 내지 변경은 당업자에 의하여 용이하게 실시될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다.
Hereinafter, the present invention will be described more specifically by way of examples. It should be understood, however, that the following examples are illustrative of the present invention only and do not limit the scope of the present invention. That is, a simple modification or change of the present invention can be easily performed by those skilled in the art, and all such modifications and alterations can be considered to be included in the scope of the present invention.
비교예 1 : 세포배양 실험 1 (신장 근위세뇨관 상피세포)Comparative Example 1: Cell culture experiment 1 (renal proximal tubular epithelial cell)
인간 신장 근위세뇨관 상피세포 (RPTEC)는 Lonza (Walkersvillle, MD, USA)에서 구입하여 배양하였다. RPTEC는 0.5% 소태아혈청(Fetal Bovine Serum, FBS)과 2mM 글루타민(Glutamine), 100 U/ml 페니실린(Penicillin), 100 mg/ml 스트렙토마이신(Streptomycin)이 첨가되어 있는 DMEM(Dulbecco's Modified Eagle's Medium, Sigma Co., St. Louis, MO, USA) 배지와 Nutrient Mixture F-12가 1:1로 함유된 배지에 5 g/ml insulin, 5 g/ml hydrocortisone, 0.5 g/ml epinephrine, 10 g/ml transferrin, 3 ng/ml human EGF and 6.5 ng/ml triiodothyronine를 첨가하여 사용하였으며 37, 5% 이산화탄소(CO2) 조건에서 배양하였다.
Human kidney proximal tubular epithelial cells (RPTEC) were purchased from Lonza (Walkersville, MD, USA) and cultured. RPTEC was prepared by adding Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 0.5% Fetal Bovine Serum (FBS), 2 mM glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin, 5 g / ml insulin, 5 g / ml hydrocortisone, 0.5 g / ml epinephrine and 10 g / ml insulin were added to the medium containing 1: 1 Nutrient Mixture F-12 transferrin, 3 ng / ml human EGF, and 6.5 ng / ml triiodothyronine. The cells were cultured under 37% CO 2 (CO 2 ) conditions.
실시예 1 : 세포배양 실험 2 (고혈당으로 인한 신세뇨관 섬유화)Example 1: Cell culture experiment 2 (renal tubular fibrosis due to hyperglycemia)
만성 고혈당으로 인한 신장섬유증 및 신세뇨관 경화증과 같은 상황을 유도하기 위하여 인간 신장 근위세뇨관 상피세포 (RPTEC)를 33mM 글루코오스(Glucose)를 첨가한 배지에서 배양하고 크리신(chrysin)을 농도별(1M-20M)로 처리하여 함께 배양하였다. 또한 삼투 대조군으로 5.5mM 글루코오스(Glucose)와 27.5mM 마니톨(Mannitol)이 함유된 배지에서 신장 근위세뇨관 상피세포를 3일간 배양하였다.
Human kidney proximal tubular epithelial cells (RPTEC) were cultured in a medium supplemented with 33 mM glucose and chrysin was added to the culture medium (1M-1) for concentration to induce conditions such as renal fibrosis and renal tubular sclerosis due to chronic hyperglycemia. 20M) and cultured together. In addition, kidney proximal tubular epithelial cells were cultured for 3 days in a medium containing 5.5 mM glucose and 27.5 mM mannitol as an osmotic control group.
실시예 2 : 결합조직 성장인자(Connetive Tissue Growth Factor, CTGF) 발현 및 분비 억제 Example 2: Expression of Connetive Tissue Growth Factor (CTGF) and inhibition of secretion
<웨스턴 블롯><Western blot>
인간 신장 근위세뇨관 상피세포 (RPTEC)의 CTGF의 발현 정도를 측정하기 위해 상기 비교예 1, 실시예 1에서 배양처리된 근위세뇨관 상피세포 (RPTEC)를 1% -Mercaproethanol, 1M -Glycerophosphate, 0.1M Na3VO4, 0.5M 불화소다(NaF)와 Protease Inhibitor Cocktail을 포함한 완충액(Buffer)으로 분해(Lysis)시켜 3000rpm에서 10분간 원심분리하여 얻은 상층액을 이용하였다. 이렇게 얻은 상층액을 10% SDS-PAGE 겔(Gel)상에서 70V로 전기영동 하였다. 겔(Gel)상에 있는 단백질은 니트로셀룰로우스 막(Nitrocellulose Membrane)으로 전이시킨 다음, 비특이적인 결합을 방지하기 위하여 3% 소혈청 알부민 (Bovine Serum Albumin, BSA)에 3시간 동안 블로킹하였다. CTGF의 일차항체(Polyclonal Rabbit Anti-human Antibody, Peprotech, Rocky Hill, NJ, USA)로 니트로셀룰로우스 막(Nitrocellulose membrane)과 교반하면서 3시간 배양한 후, 4℃에서 하룻밤 배양하고 다시 실온에서 3시간 배양한 후 이차항체인 Goat Anti-rabbit Horseradish Peroxidase (Jackson ImmunoResearch Lab., West Grove, PA, USA)로 1시간 동안 배양하였다. 니트로셀룰로스 막(Nitrocellulose Membrane)에 전이된 단백질은 Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore Corp., Billerica, MA, USA)로 검출하여 비교예 1에서 배양 처리된 정상 근위세뇨관 상피세포 (RPTEC)와 실시예 1에서 크리신(chrysin)을 1M, 10M, 20M 로 각각 처리한 세포 샘플을 X-ray 필름으로 촬영하였다.
In order to measure the expression level of CTGF in human renal proximal tubular epithelial cells (RPTEC), proximal tubular epithelial cells (RPTEC) cultured in Comparative Example 1 and Example 1 were cultured in 1% -Mercaproethanol, 1M-glycerophosphate, 0.1M Na The supernatant obtained by centrifugation at 3,000 rpm for 10 minutes was used for lysis with 3 VO 4 , a buffer containing 0.5 M sodium fluoride (NaF) and Protease Inhibitor Cocktail. The supernatant thus obtained was electrophoresed on a 10% SDS-PAGE gel at 70V. The protein on the gel was transferred to a nitrocellulose membrane and then blocked with 3% bovine serum albumin (BSA) for 3 hours to prevent nonspecific binding. After incubation for 3 hours with a nitrocellulose membrane with a primary antibody of CTGF (Polyclonal Rabbit Anti-human Antibody, Peprotech, Rocky Hill, NJ, USA), the cells were cultured overnight at 4 ° C, After incubation, the cells were incubated with a secondary antibody, Goat Anti-rabbit Horseradish Peroxidase (Jackson ImmunoResearch Lab., West Grove, PA, USA) for 1 hour. Proteins transferred to nitrocellulose membranes were detected with Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore Corp., Billerica, Mass., USA), and cultured normal normal tubular epithelial cells (RPTEC) 1 cells were treated with chrysin at 1M, 10M and 20M, respectively.
위 결과는 도 1에 나타내었다. The above results are shown in Fig.
도 1에 도시된 바와 같이 실시예 1의 삼투 대조군인 근위세뇨관 상피세포(5.5mM glucose, 27.5mM mannitol 대조군)는 CTGF의 발현 및 분비가 적었으나, 3일간 고혈당에 노출된 근위세뇨관 상피세포(RPTEC)는 CTGF의 발현 및 분비가 현저하게 증가하였다. As shown in Fig. 1, proximal tubular epithelial cells (5.5 mM glucose, 27.5 mM mannitol control), which was the osmotic control of Example 1, showed less CTGF expression and secretion than proximal tubular epithelial cells (RPTEC ) Significantly increased the expression and secretion of CTGF.
여기서, 3일간 고혈당에 노출된 RPTEC에 크리신을 1M 처리하였을 때 CTGF의 발현 및 분비에는 거의 변화가 없었으며, 크리신(chrysin)을 10M 이상 처리하였을 때는 CTGF의 발현 및 분비가 점차 억제되는 것으로 나타났다.Here, when 1M of chrysin was treated with RPTEC exposed to high glucose for 3 days, there was almost no change in the expression and secretion of CTGF, and when the chrysin was treated with 10M or more, the expression and secretion of CTGF were gradually suppressed .
따라서, 만성 고혈당에 노출된 RPTEC에 크리신(chrysin)을 10M 이상으로 처리하였을 경우에 신장 근위세뇨관 상피세포에서의 결합조직 성장인자의 생성을 억제시키는 효과가 뛰어나는 것을 알 수 있다.
Therefore, RPTEC exposed to chronic hyperglycemia is more effective in inhibiting the formation of connective tissue growth factor in proximal tubular epithelial cells when treated with chrysin at 10M or more.
실시예 3 : 제 4형 콜라겐 생성 감소Example 3: Reduction of type 4 collagen production
<웨스턴 블롯><Western blot>
인간 신장 근위세뇨관 상피세포 (RPTEC)의 제4형 콜라겐의 생성 정도를 측정하기 위해 상기 비교예 1, 실시예 1에서 배양 처리된 인간 신장 근위세뇨관 상피세포 (RPTEC)에 분해 완충액(Lysis Buffer)을 처리하여 세포 추출물을 준비하였다. 세포 추출물의 단백질 분리는 6% SDS-PAGE로 수행되었다. 전기 영동된 단백질 밴드들은 니트로셀룰로스 막(Nitrocellulose Membrane)으로 전이시키고, 비특이적인 결합을 방지하기 위하여 5% 탈지분유를 처리한 다음 제4형 콜라겐 단백질의 일차항체(Polyclonal Rabbit Anti-human Antibody, Santa Cruz Biotech., Santa Cruz, CA, USA)를 1000배로 희석하여 교반하면서 반응시키고, Goat Anti-rabbit Horseradish Peroxidase (1:10000, Jackson ImmunoReasearch Lab.)의 이차항체로 1시간 반응시켰다. 니트로셀룰로스 막(Nitrocellulose Membrane)에 있는 제 4형 콜라겐 단백질은 Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore Corp., Billerica, MA, USA)로 검출하여 비교예 1에서 배양 처리된 정상 근위세뇨관 상피세포 (RPTEC)와 실시예 1에서 크리신(chrysin)을 1M, 10M, 20M로 각각 처리한 세포 샘플을 X-ray 필름으로 촬영하였다.
To measure the degree of formation of type-4 collagen in human renal proximal tubular epithelial cells (RPTEC), a lysis buffer (Lysis Buffer) was added to the human renal proximal tubular epithelial cells (RPTEC) cultured in the above Comparative Example 1 and Example 1 To prepare a cell extract. Protein separation of cell extracts was performed by 6% SDS-PAGE. The electrophoretic protein bands were transferred to a nitrocellulose membrane (Nitrocellulose Membrane), treated with 5% defatted milk powder to prevent nonspecific binding, and incubated with primary antibody of the type-4 collagen protein (Polyclonal Rabbit Anti-human Antibody, Santa Cruz (1: 10000, Jackson ImmunoReasearch Lab.) For 1 hour. The reaction mixture was diluted 1: 1000 with 1: 10000 dilution of Biotec, Santa Cruz, Calif. The type IV collagen protein in the nitrocellulose membrane was detected with Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore Corp., Billerica, Mass., USA), and the normal proximal tubular epithelial cells (RPTEC) And chrysin at 1M, 10M, and 20M in Example 1, respectively, were photographed with X-ray film.
위 결과는 도 2에 나타내었다. The above results are shown in Fig.
도 2에 도시된 바와 같이 실시예 1의 삼투 대조군인 근위세뇨관 상피세포(5.5mM glucose, 27.5mM mannitol 대조군)는 4형 콜라겐 분비 생성이 적었으나, 3일간 고혈당에 노출된 근위세뇨관 상피세포(RPTEC)는 4형 콜라겐 분비가 현저하게 증가하였다.As shown in FIG. 2, proximal tubular epithelial cells (5.5 mM glucose, 27.5 mM mannitol control), which was the osmotic control of Example 1, produced less progenitor collagen secretion than proximal tubular epithelial cells (RPTEC ) Significantly increased the secretion of type 4 collagen.
여기서, 3일간 고혈당에 노출된 RPTEC에 크리신(chrysin)을 1M 처리하였을 때 4형 콜라겐 분비에는 거의 변화가 없었으며, 크리신(chrysin)을 10M 이상 처리하였을 때 4형 콜라겐 분비가 점차 억제되는 것으로 나타났다.Here, when 1 M of chrysin was treated with RPTEC exposed to high glucose for 3 days, there was almost no change in the secretion of type 4 collagen, and when the chrysin was treated with 10 M or more, the type 4 collagen secretion was gradually suppressed Respectively.
따라서, 만성 고혈당에 노출된 RPTEC에 크리신을 10M 이상으로 처리하였을 경우에 신장 근위세뇨관 상피세포에서 결합조직을 형성하는 콜라겐의 분비 및 축적을 억제하는 데에 뛰어난 효과를 발휘하는 것을 알 수 있다.
Therefore, it can be seen that when RPTEC exposed to chronic hyperglycemia is treated with chrysin at 10M or more, it exerts an excellent effect in inhibiting the secretion and accumulation of collagen forming connective tissue in proximal tubular epithelium of kidney.
실시예 4 : 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT) 관련 단백질인 N-cadherin의 발현 감소 및 EMT를 억제하는 세포부착 단백질인 E-cadherin의 발현 증가Example 4: Reduction of expression of N-cadherin, an epithelial-mesenchymal transition (EMT) -related protein, and increase of expression of E-cadherin, a cell adhesion protein that inhibits EMT
<웨스턴 블롯><Western blot>
인간 신장 근위세뇨관 상피세포 (RPTEC)의 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT) 관련 단백질인 N-cadherin의 발현 및 EMT를 억제하는 세포부착 단백질인 E-cadherin의 발현 정도를 측정하기 위해 상기 비교예 1, 실시예 1에서 배양 처리된 인간 신장 근위세뇨관 상피세포 (RPTEC)에 분해 완충액(Lysis Buffer)을 처리하여 세포 추출물을 준비하였다. 세포 추출물의 단백질 분리는 8% SDS-PAGE로 수행되었다. 전기 영동된 단백질 밴드들은 니트로셀룰로스 막(Nitrocellulose Membrane)으로 전이시키고, 비특이적인 결합을 방지하기 위하여 5% 탈지분유를 처리한 다음 N-cadherin 단백질의 일차항체(Polyclonal Rabbit Anti-human Antibody, Cambridge, UK)와 E-cadherin 단백질의 일차항체(Polyclonal Rabbit Anti-human Antibody, Cell signaling Technology, CA, USA)를 1000배로 희석하여 교반하면서 반응시키고, Goat Anti-rabbit Horseradish Peroxidase (1:10000, Jackson ImmunoReasearch Lab.)의 이차항체로 1시간 반응시켰다. 니트로셀룰로스 막(Nitrocellulose Membrane)에 있는 N-cadherin과 E-cadherin 단백질은 Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore Corp., Billerica, MA, USA)로 검출하여 비교예 1에서 배양 처리된 정상 근위세뇨관 상피세포 (RPTEC)와 실시예 1에서 크리신(chrysin)을 1M, 10M, 20M로 각각 처리한 세포 샘플을 X-ray 필름으로 촬영하였다.
Expression of N-cadherin, an Epithelial-Mesenchymal Transition (EMT) -related protein of human renal proximal tubular epithelial cells (RPTEC), and the expression level of E-cadherin, a cell adhesion protein that inhibits EMT The human renal proximal tubular epithelial cells (RPTEC) cultured in the above Comparative Example 1 and Example 1 were treated with a lysis buffer to prepare a cell extract. Protein separation of cell extracts was performed by 8% SDS-PAGE. The electrophoretic protein bands were transferred to a nitrocellulose membrane (Nitrocellulose Membrane), treated with 5% skimmed milk powder to prevent nonspecific binding, and incubated with primary antibodies of the N-cadherin protein (Polyclonal Rabbit Anti-Human Antibody, Cambridge, UK ) And E-cadherin protein primary antibody (Polyclonal Rabbit Anti-Human Antibody, Cell Signaling Technology, CA, USA) were diluted 1000-fold and reacted with agitation. Goat anti-rabbit Horseradish peroxidase (1: 10000, Jackson ImmunoReasearch Lab. ) Secondary antibody for 1 hour. The N-cadherin and E-cadherin proteins in the nitrocellulose membrane were detected with Immobilon western chemiluminescent horseradish peroxidase substrate (Millipore Corp., Billerica, MA, USA), and the normal proximal tubular epithelial cells cultured in Comparative Example 1 (RPTEC) and a cell sample treated with 1M, 10M, and 20M of chrysin in Example 1, respectively, were taken with X-ray film.
위 결과는 도 3에 나타내었다. The above results are shown in Fig.
도 3에 도시된 바와 같이 실시예 1의 삼투 대조군인 근위세뇨관 상피세포(5.5mM glucose, 27.5mM mannitol 대조군)는 EMT 관련 단백질 마커인 N-cadherin의 발현량은 대조군에서는 발현량이 적었으나, 3일간 고혈당에 노출된 근위세뇨관 상피세포(RPTEC)에서는 N-cadherin의 발현량이 현저하게 증가하였다. 여기에 3일간 고혈당에 노출된 RPTEC에 크리신(chrysin)을 1M 처리하였을 때 N-cadherin의 발현량에는 거의 변화가 없었으며, 크리신을 10M 이상 처리하였을 때 N-cadherin의 발현량이 농도 의존적으로 점차 억제되는 것으로 나타났다. 반면에 EMT를 억제하는 세포부착 단백질인 E-cadherin의 발현은 3일간 고혈당에 노출된 근위세뇨관 상피세포(RPTEC)에서 현저하게 감소하였다. 여기에 크리신을 농도별로 처리한 경우 농도 의존적으로 생성이 증가하는 경향을 보였다.As shown in FIG. 3, the expression level of N-cadherin, an EMT-related protein marker, in the proximal tubular epithelial cells (5.5 mM glucose, 27.5 mM mannitol control) of Example 1 was small in the control group, The expression of N-cadherin was significantly increased in proximal tubular epithelial cells (RPTEC) exposed to hyperglycemia. There was no change in the expression level of N-cadherin when 1M of chrysin was treated with RPTEC exposed to hyperglycemia for 3 days, and the expression level of N-cadherin gradually increased gradually when the chrysin was treated with 10M or more Respectively. On the other hand, the expression of E-cadherin, a cell attachment protein that suppresses EMT, was significantly decreased in proximal tubular epithelial cells (RPTEC) exposed to high glucose for 3 days. When the concentration of chrysin was treated at different concentrations, the production was increased in a concentration dependent manner.
따라서, 만성 고혈당에 노출된 RPTEC에 크리신(chrysin)을 10M 이상으로 처리하였을 경우에 신장 근위세뇨관 상피세포에서 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT) 관련 단백질인 N-cadherin의 발현을 감소시키고, EMT를 억제하는 세포부착 단백질인 E-cadherin의 발현을 증가시키는 데에 뛰어난 효과를 발휘하는 것을 알 수 있다.Therefore, the expression of N-cadherin, an epithelial-mesenchymal transition (EMT) -related protein, in renal proximal tubular epithelial cells when treated with chrysin at a concentration of 10M or more in RPTEC exposed to chronic hyperglycemia And E-cadherin, which is a cell attachment protein that suppresses EMT, and thus exerts an excellent effect in increasing the expression of E-cadherin.
Claims (3)
만성 고혈당에 노출된 신장 근위세뇨관 상피세포 (RPTEC)의 결합조직 성장인자(CTGF) 분비를 억제하고, 제 4형 콜라겐 생성을 감소시키는 것을 특징으로 하는 크리신(chrysin)을 함유하는 당뇨합병성 신세뇨관 섬유화로 인한 신장섬유증 개선용 건강기능성 식품 조성물.The method according to claim 1,
A method of inhibiting the secretion of connective tissue growth factor (CTGF) of renal proximal tubular epithelial cells (RPTEC) exposed to chronic hyperglycemia and reducing the production of type-IV collagen, comprising administering a chrysin- A health functional food composition for improving renal fibrosis caused by tubular fibrosis.
만성 고혈당에 노출된 신장 근위세뇨관 상피세포 (RPTEC)의 상피-중간엽세포 이행(Epithelial-Mesenchymal Transition, EMT) 관련 단백질 마커인 N-카데린의 발현을 감소시키고, EMT를 억제하는 세포부착단백질인 E-카데린의 발현을 증가시켜 상피-중간엽세포 이행을 억제하는 것을 특징으로 하는 크리신(chrysin)을 함유하는 당뇨합병성 신세뇨관 섬유화로 인한 신장섬유증 개선용 건강기능성 식품 조성물.The method according to claim 1,
(RPTEC), which is a cell attachment protein that suppresses the expression of N-cadherin, which is an epithelial-mesenchymal transition (EMT) -related protein marker, in the kidney proximal tubular epithelium (RPTEC) exposed to chronic hyperglycemia The present invention relates to a health functional food composition for improving renal fibrosis caused by diabetic complicated renal tubular fibrosis containing chrysin, characterized by inhibiting epithelial-mesenchymal cell migration by increasing E-cadherin expression.
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| KR101949471B1 (en) | 2018-10-08 | 2019-02-18 | 한림대학교 산학협력단 | Composition of improvement, prevention and treatment in renal fibrosis or cirrhosis of the kidney glomerulus or albuminuria |
| WO2019151732A1 (en) * | 2018-01-31 | 2019-08-08 | 한림대학교 산학협력단 | Pharmaceutical composition for preventing or treating diabetes complications comprising novel chrysin derivative compound as active ingredient |
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| JP2004516257A (en) | 2000-12-21 | 2004-06-03 | ザ キグリー コーポレーション | Compositions and methods for treating diabetic neuropathy |
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