KR20230085385A - Pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells comprising bifenthrin - Google Patents
Pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells comprising bifenthrin Download PDFInfo
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- KR20230085385A KR20230085385A KR1020210173492A KR20210173492A KR20230085385A KR 20230085385 A KR20230085385 A KR 20230085385A KR 1020210173492 A KR1020210173492 A KR 1020210173492A KR 20210173492 A KR20210173492 A KR 20210173492A KR 20230085385 A KR20230085385 A KR 20230085385A
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- cells
- bifenthrin
- endometrial
- trophoblast
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Abstract
본 발명은 비펜트린을 유효성분으로 포함하는 조성물에 관한 것으로, 보다 상세하게는 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물, 및 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.
본 발명에 따른 비펜트린을 유효성분으로 포함하는 조성물은 자궁내막 세포 또는 영양막 세포 내 PI3K/AKT, MAPK 세포전달 메커니즘과 그 하위 신호 전달물질의 인산화를 다양하게 조절하고, 산화스트레스 조절에 관여하는 소포체 스트레스 반응 인자의 발현을 증가시킴으로써 자궁내막 세포 및 영양막 세포의 증식을 억제하고, 세포 사멸을 유도하는 효과가 있는바, 자궁내막 세포 또는 영양막 세포의 비정상적 증식과 관련된 질환을 예방 및 치료할 수 있는 의약품 및 기능성 식품과 관련된 분야에서 유용하게 사용될 수 있다.The present invention relates to a composition containing bifenthrin as an active ingredient, and more particularly, to a pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells containing bifenthrin as an active ingredient; and a health functional food composition for preventing or improving abnormal proliferation-related diseases of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.
The composition containing bifenthrin as an active ingredient according to the present invention variously regulates the phosphorylation of PI3K/AKT and MAPK cell transduction mechanisms and sub-signal transmitters in endometrial cells or trophoblast cells, and is involved in oxidative stress regulation. By increasing the expression of endoplasmic reticulum stress response factors, it inhibits proliferation of endometrial cells and trophoblast cells and induces apoptosis, thereby preventing and treating diseases associated with abnormal proliferation of endometrial cells or trophoblast cells. And it can be usefully used in fields related to functional foods.
Description
본 발명은 비펜트린을 유효성분으로 포함하는 조성물에 관한 것으로, 보다 상세하게는 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물, 및 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.The present invention relates to a composition containing bifenthrin as an active ingredient, and more particularly, to a pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells containing bifenthrin as an active ingredient; and a health functional food composition for preventing or improving abnormal proliferation-related diseases of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.
자궁내막증, 자궁내막증식증, 자궁내막 폴립, 자궁내막암과 같은 질환은 자궁내막 세포의 비정상적인 증식에 의해 야기되는 질환이다. 그 중, 자궁내막증은 가임기 여성의 난소, 난관, 복벽등에 자궁내막 세포가 비정상적으로 증식하여 35-50% 환자에게서 불임을 야기하는 질환으로서(Vigano et al, Best Pract Res Clin Obstet Gynaecol, 2004; Bulun, N Engl J Med, 2009), 이에 대한 병리학적 기전에 대한 연구가 활발하게 수행되고 있으나, 아직까지 정확한 발병 원인은 밝혀지지 않고 있다. 자궁내막증의 기본적인 치료 방법으로는 수술적 제거와 호르몬치료 방법이 있으며, 자궁내막증은 예후 인자를 기반으로 한 진단이 아닌, 증상 위주의 진단이 이루어지기 때문에 수술적 제거를 진행하여도 5년 내 재발률이 30-40%에 육박하는 것으로 알려져 있다(Busacca et al., J Obstet Gynecol, 2006: Vignali et al., J Minim Invasive Gynecol, 2005). 또한, 자궁내막암은 자궁 조직의 과잉증식에 의한 악성종양으로 평균 출산 연령의 증가 및 생활 습관의 변화로 인하여 발병이 지속적으로 증가하는 추세이며, 미국에서 발병률이 가장 높은 부인과 종양으로 알려져 있다(Mills, A.M. and T.A. Longacre, Semin Diagn Pathol, 2010).Diseases such as endometriosis, endometrial hyperplasia, endometrial polyp, and endometrial cancer are diseases caused by abnormal proliferation of endometrial cells. Among them, endometriosis is a disease in which endometrial cells abnormally proliferate in the ovaries, fallopian tubes, and abdominal wall of women of childbearing age, causing infertility in 35-50% of patients (Vigano et al, Best Pract Res Clin Obstet Gynaecol, 2004; Bulun , N Engl J Med, 2009), and studies on the pathological mechanism thereof are being actively conducted, but the exact cause of the disease has not yet been identified. The basic treatment methods for endometriosis include surgical removal and hormonal therapy, and because endometriosis is diagnosed based on symptoms rather than prognostic factors, the recurrence rate within 5 years is low even after surgical removal. is known to approach 30-40% (Busacca et al., J Obstet Gynecol, 2006: Vignali et al., J Minim Invasive Gynecol, 2005). In addition, endometrial cancer is a malignant tumor caused by overgrowth of uterine tissue, and its incidence is continuously increasing due to the increase in the average age of childbirth and changes in lifestyle, and is known as a gynecological tumor with the highest incidence in the United States (Mills , A. M. and T. A. Longacre, Semin Diagn Pathol, 2010).
한편, 융모막암, 포상기태, 융모선종와 같은 임신성 영양막 질환은 영양막 세포의 비정상적인 증식, 과도한 이동성과 침투성으로 인해 발병하는 질병이다. 특히 융모막암은 임신성 영양막 질환중 가장 악성의 질병으로 주로 임신 초기에 포상기태로부터 발병하는 경우가 대다수이며(VCMak et al, Carcinogenesis, 2016; LDuffy et al, Journal of clinical medicine research, 2015), 영양막 조직에서 유래된 악성종양인 융모막암은 임신기에 20,000~40,000 명 당 1명의 비율로 나타나는 것으로 보고된바 있다(Soper et al, Gynecol Oncol, 2004). 또한, 융모막암은 혈관침투성이 매우 높으며 폐나 질 같은 타기관으로의 전이가 빈번하게 일어나는 것으로 알려져있다 (Geramizadeh and Rad, Indian J Pathol Microbiol, 2012). 일반적인 융모막암의 치료 방법은 EMA-CO라고 불리는 화학적치료법을 실시하는 것이다. 하지만, 융모막암 환자의 15-25%는 이러한 화학적 치료법에 대해 저항성 및 나쁜 예후를 나타낸다(Powles et al, Br J Cancer, 2007). 따라서 융모막암에 나타나는 항암제 내성을 극복하기 위해 더욱 효과적인 치료제 개발을 통한 접근법이 필요한 실정이다.On the other hand, gestational trophoblast diseases such as chorionic carcinoma, molar molars, and choriocarcinoma are diseases caused by abnormal proliferation, excessive mobility and permeability of trophoblast cells. In particular, chorionic carcinoma is the most malignant disease among gestational trophoblast diseases, and most cases arise from molar follicles in the early stages of pregnancy (VCMak et al, Carcinogenesis, 2016; LDuffy et al, Journal of clinical medicine research, 2015), and trophoblast tissue Choriocarcinoma, a malignant tumor derived from , has been reported to occur at a rate of 1 per 20,000 to 40,000 during pregnancy (Soper et al, Gynecol Oncol, 2004). In addition, it is known that choriocarcinoma has very high vascular permeability and metastases to other organs such as the lung or vagina frequently occur (Geramizadeh and Rad, Indian J Pathol Microbiol, 2012). A common treatment for choriocarcinoma is to administer chemotherapy called EMA-CO. However, 15-25% of choriocarcinoma patients are resistant to these chemotherapy and show poor prognosis (Powles et al, Br J Cancer, 2007). Therefore, in order to overcome anticancer drug resistance in choriocarcinoma, there is a need for an approach through the development of more effective therapeutic agents.
이처럼 자궁내막 세포 또는 영양막 세포의 비정상적 증식은 다양한 심각한 질환을 야기하며, 이들 질환은 모두 종래 치료법의 한계로 인하여 새로운 치료제의 개발이 요구되고 있다. As such, abnormal proliferation of endometrial cells or trophoblast cells causes various serious diseases, and all of these diseases require the development of new therapeutic agents due to the limitations of conventional treatments.
전술한 기술적 배경하에서, 본 발명자들은 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환 치료를 위한 새로운 물질을 개발하기 위해 예의 노력한 결과, 비펜트린(bifenthrin)이 자궁내막 세포 또는 영양막 세포 내 PI3K/AKT 및 MAPK 신호전달기전의 활성을 조절하여 자궁내막 세포 또는 영양막 세포의 증식과 이주성 및 침투성을 억제하고, 세포 사멸을 유도한다는 것을 확인함으로써 본 발명을 완성하였다.Under the aforementioned technical background, the present inventors have made diligent efforts to develop new substances for the treatment of abnormal proliferation-related diseases of endometrial cells or trophoblast cells. The present invention was completed by confirming that proliferation, migration, and invasiveness of endometrial cells or trophoblast cells were inhibited, and cell death was induced by regulating the activity of the MAPK signaling pathway.
본 발명은 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases associated with abnormal proliferation of endometrial cells or trophoblasts, containing bifenthrin as an active ingredient.
또한, 본 발명은 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a health functional food composition for preventing or improving diseases related to abnormal proliferation of endometrial cells or trophoblast cells, containing bifenthrin as an active ingredient.
또한, 본 발명은 비펜트린을 유효성분으로 포함하는 조성물을 이용하여 자궁내막 세포 또는 영양막 세포의 증식, 이주 및 침투 능력을 억제하는 방법을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a method for inhibiting proliferation, migration and penetration of endometrial cells or trophoblasts by using a composition containing bifenthrin as an active ingredient.
또한, 본 발명은 비펜트린을 유효성분으로 포함하는 조성물을 이용하여 자궁내막 세포 또는 영양막 세포의 사멸 효과를 향상시키는 방법을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a method for enhancing the killing effect of endometrial cells or trophoblast cells by using a composition containing bifenthrin as an active ingredient.
본 발명은 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating diseases associated with abnormal proliferation of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.
또한, 본 발명은 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving diseases related to abnormal proliferation of endometrial cells or trophoblasts, containing bifenthrin as an active ingredient.
또한, 본 발명은 비펜트린을 유효성분으로 포함하는 조성물을 이용하여 자궁내막 세포 또는 영양막 세포의 증식, 이주 및 침투 능력을 억제하는 방법을 제공한다.In addition, the present invention provides a method for inhibiting proliferation, migration and penetration of endometrial cells or trophoblasts by using a composition containing bifenthrin as an active ingredient.
또한, 본 발명은 비펜트린을 유효성분으로 포함하는 조성물을 이용하여 자궁내막 세포 또는 영양막 세포의 사멸 효과를 향상시키는 방법을 제공한다.In addition, the present invention provides a method for enhancing the killing effect of endometrial cells or trophoblast cells by using a composition containing bifenthrin as an active ingredient.
본 발명에 따른 비펜트린을 유효성분으로 포함하는 조성물은 자궁내막 세포 또는 영양막 세포 내 PI3K/AKT, MAPK 세포전달 메커니즘과 그 하위 신호 전달물질의 인산화를 다양하게 조절하고, 산화스트레스 조절에 관여하는 소포체 스트레스 반응 인자의 발현을 증가시킴으로써 자궁내막 세포 및 영양막 세포의 증식을 억제하고, 세포 사멸을 유도하는 효과가 있는바, 자궁내막 세포 또는 영양막 세포의 비정상적 증식과 관련된 질환을 예방 및 치료할 수 있는 의약품 및 기능성 식품과 관련된 분야에서 유용하게 사용될 수 있다.The composition containing bifenthrin as an active ingredient according to the present invention variously regulates the phosphorylation of PI3K/AKT and MAPK cell transduction mechanisms and sub-signal transmitters in endometrial cells or trophoblast cells, and is involved in oxidative stress regulation. By increasing the expression of endoplasmic reticulum stress response factors, it inhibits proliferation of endometrial cells and trophoblast cells and induces apoptosis, thereby preventing and treating diseases associated with abnormal proliferation of endometrial cells or trophoblast cells. And it can be usefully used in fields related to functional foods.
도 1은 비펜트린이 돼지 영양막 세포 및 자궁내막 세포의 증식에 미치는 영향을 분석한 결과를 나타낸 것이다.
도 2는 비펜트린에 의한 돼지 영양막 세포 및 자궁내막 세포의 사멸 유도 효과를 분석한 결과를 나타낸 것이다.
도 3은 비펜트린에 의한 돼지 영양막 세포 및 자궁내막 세포의 세포주기 진행 억제 효과를 분석한 결과를 나타낸 것이다.
도 4는 비펜트린에 의한 돼지 영양막 세포 및 자궁내막 세포에서의 미토콘드리아 기능 장애 유도 효과를 분석한 결과를 나타낸 것이다.
도 5는 비펜트린에 의한 돼지 영양막 세포 및 자궁내막 세포에서의 소포체 스트레스 유도 및 칼슘 항상성 저해 효과를 분석한 결과를 나타낸 것이다.
도 6은 비펜트린이 돼지 영양막 세포 및 자궁내막 세포 내 신호전달기전에 미치는 영향을 분석한 결과를 나타낸 것이다.
도 7은 비펜트린에 의한 돼지 영양막 세포 및 자궁내막 세포의 스페로이드 형성 및 성장 저해 효과를 분석한 결과를 나타낸 것이다.
도 8은 영양막 세포 또는 자궁내막 세포 내 비펜트린에 의한 세포 증식 억제 및 세포 사멸 기전을 나타낸 것이다.1 shows the results of analyzing the effect of bifenthrin on the proliferation of porcine trophoblast and endometrial cells.
Figure 2 shows the results of analyzing the effect of inducing death of porcine trophoblast and endometrial cells by bifenthrin.
Figure 3 shows the results of analyzing the cell cycle progression inhibition effect of porcine trophoblast cells and endometrial cells by bifenthrin.
Figure 4 shows the results of analyzing the effect of inducing mitochondrial dysfunction in porcine trophoblast cells and endometrial cells by bifenthrin.
5 shows the results of analyzing the effects of bifenthrin on inducing endoplasmic reticulum stress and inhibiting calcium homeostasis in porcine trophoblast cells and endometrial cells.
Figure 6 shows the results of analyzing the effect of bifenthrin on signal transduction mechanisms in porcine trophoblast cells and endometrial cells.
Figure 7 shows the results of analyzing the effect of bifenthrin on spheroid formation and growth inhibition of porcine trophoblast and endometrial cells.
8 shows the mechanism of inhibition of cell proliferation and cell death by bifenthrin in trophoblast or endometrial cells.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.
본 발명에서는 비펜트린의 영양막 세포 또는 자궁내막 세포의 증식 억제 및 세포 사멸 기전을 밝혔다(도 8).In the present invention, the mechanism of bifenthrin's inhibition of trophoblast or endometrial cell proliferation and apoptosis was found (FIG. 8).
본 발명은 일 관점에서 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating diseases associated with abnormal proliferation of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.
본 발명은 다른 관점에서 비펜트린을 유효성분으로 포함하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.In another aspect, the present invention relates to a functional health food composition for preventing or improving diseases related to abnormal proliferation of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.
본 발명에서 사용된 용어 "조성물"은 특정 성분을 포함하는 산물뿐만 아니라, 특정 성분의 배합에 의해 직접 또는 간접적으로 만들어지는 임의의 산물을 포함하는 것으로 간주된다.As used herein, the term "composition" is considered to include not only products containing specific components, but also any products made directly or indirectly by combining specific components.
본 발명에 있어서, 상기 영양막 세포 또는 자궁내막 상피세포는 포유동물 유래의 세포일 수 있으며, 상기 포유동물은 설치목(예를 들어, 생쥐, 쥐, 햄스터, 게르빌루스 및 기니피그), 우제목(예를 들어, 소, 양, 돼지, 염소, 사슴, 기린 및 영양), 기제목(예를 들어, 말, 당나귀, 코뿔소 및 맥), 식육목(예를 들어, 개, 고양이, 호랑이, 늑대, 여우, 사자, 치타, 표범, 너구리, 오소리, 퓨마, 재규어 및 살쾡이), 토끼목(토끼 및 우는 토끼), 식충목(예를 들어, 고슴도치, 두더지 및 솔레노돈) 및 영장목(예를 들어, 침팬지, 오랑우탄, 고릴라, 보노보, 일본원숭이, 붉은털원숭이)일 수 있다.In the present invention, the trophoblast cells or endometrial epithelial cells may be mammalian-derived cells, and the mammals are rodents (eg, mice, rats, hamsters, gerbils and guinea pigs), corticosteroids ( e.g. cattle, sheep, pigs, goats, deer, giraffes and antelopes), branch orders (e.g. horses, donkeys, rhinos and tapirs), carnivores (e.g. dogs, cats, tigers, wolves, foxes) , lions, cheetahs, leopards, raccoons, badgers, pumas, jaguars and bobcats), lagomorphs (rabbits and songbirds), insectivores (e.g. hedgehogs, moles and solenodons) and primates (e.g. chimpanzees, orangutans, gorillas, bonobos, Japanese macaques, rhesus monkeys).
본 발명에 있어서, 약학적 조성물은 약제학적으로 허용가능한 담체를 함유하는 것을 특징으로 할 수 있고, 상기 담체는 이온 교환 수지, 알루미나, 알루미늄 스테아레이트, 레시틴, 혈청 단백질, 완충 물질, 물, 염, 전해질, 교질성 실리카, 마그네슘 트리실리케이트, 폴리비닐피롤리돈, 셀룰로즈계 기질, 폴리에틸렌 글리콜, 나트륨 카르복시메틸셀룰로즈, 폴리아릴레이트, 왁스, 폴리에틸렌 글리콜 및 양모지로 구성된 군에서 선택되는 하나 이상인 것을 특징으로 할 수 있다.In the present invention, the pharmaceutical composition may be characterized by containing a pharmaceutically acceptable carrier, and the carrier may include ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer substances, water, salt, At least one selected from the group consisting of electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and wool. can
본 발명에 있어서, 약학적 조성물은 정맥내, 복강내, 근육내, 동맥내, 구강, 심장내, 골수내, 경막내, 경피, 장관, 피하, 설하 또는 국부 투여용으로 제형화하는 것을 특징으로 할 수 있고, 완충제, 항균성 보존제, 계면활성제, 산화방지제, 긴장성 조정제, 방부제, 증점제 및 점도 개질제로 구성된 군에서 선택된 어느 하나 이상의 보조제를 추가로 함유하는 것을 특징으로 할 수 있으며, 용액, 현탁액, 에멀젼, 겔 및 분말로 구성된 군에서 선택되는 제형을 가지는 것을 특징으로 할 수 있다.In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, buccal, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It may be characterized in that it further contains at least one adjuvant selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity adjusters, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized by having a formulation selected from the group consisting of gels and powders.
본 발명의 약학적 조성물의 적합한 투여량은 증상의 경중도, 환자의 체중, 연령, 성, 투여 방식 및 투여시간 등과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정할 수 있다.A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, the patient's weight, age, sex, administration method and administration time, and usually a skilled physician is effective in the treatment or prevention desired. The dosage can be easily determined.
본 발명에 있어서, 건강기능식품은 건강기능식품에 관한 법률 제6722호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 식품을 의미한다.In the present invention, health functional food refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body according to Act No. It refers to food consumed for the purpose of obtaining useful effects for health purposes such as regulation or physiological action.
본 발명의 건강기능식품 조성물은 당해 기술분야에 공지되어 있는 통상적인 건강기능식품의 제형으로 제제화될 수 있고, 과립제, 정제, 환제, 현탁액, 에멀젼, 시럽제, 껌, 차, 젤리, 각종 음료수, 드링크제, 알코올 음료 등으로 제조될 수 있으며, 상기 건강기능식품의 종류에는 특별한 제한이 없다.The health functional food composition of the present invention can be formulated into a conventional health functional food formulation known in the art, and can be formulated into granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, and drinks. , It can be made into alcoholic beverages, etc., and there is no particular limitation on the type of health functional food.
본 발명의 건강기능식품 조성물은 인체를 비롯한 동물 신체에 투여하기 적합한 임의의 생약 형태, 더욱 구체적으로는 경구 투여에 통상적인 임의의 형태, 예를 들어 식품 또는 사료, 식품 또는 사료의 첨가제 및 보조제, 강화된 식품 또는 사료, 정제, 환제, 과립, 캡슐 및 발포 배합물 등과 같은 고체 형태 또는 용액, 현탁액, 유화액, 음료, 페이스트 등과 같은 액체형태 일 수 있고, 영양제, 비타민, 전해질, 감미제, 착색제, 유기산, 방부제 등을 함유할 수 있으며, 이러한 성분들을 독립적으로 또는 조합하여 사용할 수 있다.The health functional food composition of the present invention is any herbal form suitable for administration to the human body or animal body, more specifically, any form conventional for oral administration, such as food or feed, food or feed additives and supplements, Solid forms such as fortified foods or feeds, tablets, pills, granules, capsules and effervescent formulations, or liquid forms such as solutions, suspensions, emulsions, beverages, pastes, etc., nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, It may contain preservatives and the like, and these components may be used independently or in combination.
본 발명에 따르면, 상기 비펜트린이 PI3K/AKT 및 MAPK 신호전달기전을 조절하는 것을 특징으로 할 수 있다. According to the present invention, the bifenthrin may be characterized in that it regulates PI3K/AKT and MAPK signaling mechanisms.
본 발명에 따르면, 상기 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환은 자궁내막 세포 또는 영양막 세포의 비정상적 발달에 의해 유발되는 것이라면 모두 가능하다. 예를 들어, 상기 자궁내막 세포의 비정상적 증식 관련 질환은 자궁내막증(endometriosis), 자궁내막증식증(endometrial hyperplasia), 자궁내막 폴립(endometrial polyp) 및 자궁내막암(endometrial cancer)으로 이루어진 군에서 선택되는 1종 이상일 수 있다. 또한, 상기 영양막 세포의 비정상적 증식 관련 질환은 임신성 영양막 질환(Gestational trophoblastic disease)일 수 있으며, 상기 임신성 영양막 질환은 융모막암(choriocarcinoma), 포상기태(hydatidiform mole) 및 융모선종(villous adenoma)로 이루어진 군에서 선택되는 1종 이상일 수 있다. According to the present invention, any disease associated with abnormal proliferation of endometrial cells or trophoblast cells is possible as long as it is caused by abnormal development of endometrial cells or trophoblast cells. For example, the disease associated with abnormal proliferation of endometrial cells is 1 selected from the group consisting of endometriosis, endometrial hyperplasia, endometrial polyp, and endometrial cancer. There may be more than one species. In addition, the abnormal proliferation-related disease of trophoblastic cells may be a gestational trophoblastic disease, and the gestational trophoblastic disease is a group consisting of choriocarcinoma, hydatidiform mole, and villous adenoma. It may be one or more selected from.
본 발명에 있어서, 상기 비펜트린이 영양막 세포 및 자궁 내막 세포 내 산화스트레스 조절에 관여하는 소포체 스트레스 반응 인자의 발현을 증가시키는 것을 특징으로 할 수 있다. In the present invention, the bifenthrin may increase the expression of endoplasmic reticulum stress response factors involved in oxidative stress regulation in trophoblast and endometrial cells.
본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와 순차적 또는 동시에 투여될 수 있다.The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, or may be administered sequentially or simultaneously with conventional therapeutic agents.
본 발명은 또 다른 관점에서, 비펜트린을 유효성분으로 포함하는 조성물을 이용하여 인간을 제외한 포유동물 유래 자궁내막 세포 또는 영양막 세포의 증식, 이주 및 침투 능력을 억제하는 방법에 관한 것이다.In another aspect, the present invention relates to a method for inhibiting proliferation, migration, and invasion of endometrial cells or trophoblast cells derived from non-human mammals by using a composition containing bifenthrin as an active ingredient.
본 발명은 또 다른 관점에서, 비펜트린을 유효성분으로 포함하는 조성물을 이용하여 인간을 제외한 포유동물 유래 자궁내막 세포 또는 영양막 세포의 사멸 효과를 향상시키는 방법에 관한 것이다.In another aspect, the present invention relates to a method for enhancing the killing effect of non-human endometrial cells or trophoblast cells by using a composition containing bifenthrin as an active ingredient.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
실험 방법Experiment method
세포배양cell culture
돼지의 영양막 세포주 (pTr)는 임신 12일차 암퇘지의 배반포에서 추출하였으며, 돼지의 자궁내막 세포주 (pLE)는 임신 12일차 암퇘지의 자궁내막에서 추출하였다. 두 세포의 단층 배양을 위하여 10% 소태아혈청과 1%의 항생제 및 0.1%의 인슐린을 첨가한 DMEM/F12 1:1 배양액에서 배양하였다. The porcine trophoblast cell line (pTr) was extracted from the blastocyst of a sow on day 12 of pregnancy, and the porcine endometrial cell line (pLE) was extracted from the endometrium of a sow on day 12 of pregnancy. For monolayer culture of the two cells, they were cultured in DMEM/F12 1:1 medium supplemented with 10% fetal bovine serum, 1% antibiotics and 0.1% insulin.
실험 재료experimental material
비펜트린(Bifnthrin)은 ChemService로부터 구매하여 사용하였으며, 세포 내 신호전달기작을 확인하기 위하여 phospho-AKT, ERK1/2, P70S6K, S6, JNK, P38, eIF2α 단백질 및 total-AKT, ERK1/2, P70S6K, S6, JNK, P38, BAX, BAK, Cytochrome C 단백질을 Cell Cignaling Technology사로부터 구매하였다. 이 외, GRP78, α-tubulin, phospho-PERK, PCNA 항체는 Santa Cruz Biotechnology사에서 구매하였다. 또한, 타겟 신호전달경로의 억제에 따른 효과를 규명하기 위하여 PI3K 억제제인 LY294002를 Cell Signaling Technology사로부터, ERK1/2, JNK, P38 억제제인 U0126, SP600125, SB203580을 Enzo Life Science사로부터 구매하여 사용하였다.Bifnthrin was purchased and used from ChemService, and phospho-AKT, ERK1/2, P70S6K, S6, JNK, P38, eIF2α proteins and total-AKT, ERK1/2, P70S6K, S6, JNK, P38, BAX, BAK, and Cytochrome C proteins were purchased from Cell Signaling Technology. In addition, GRP78, α-tubulin, phospho-PERK, and PCNA antibodies were purchased from Santa Cruz Biotechnology. In addition, to investigate the effect of inhibition of the target signaling pathway, PI3K inhibitor LY294002 was purchased from Cell Signaling Technology, and ERK1/2, JNK, and P38 inhibitors U0126, SP600125, and SB203580 were purchased from Enzo Life Science and used. .
Tetrazoliumtetrazolium bromide를 이용한 세포 독성 효과 분석 Analysis of cytotoxic effect using bromide
비펜트린의 세포 독성을 평가하기 위하여 96well에 배양한 pTr, pLE 세포에 비펜트린을 처리한 뒤, 48시간 배양하였다. 그리고 10 μL의 MTT labeling reagent를 추가로 분주한 뒤, 37℃에서 4시간 동안 인큐베이션하였다. 각각의 well에 solubilization solution을 100 μL씩 분주하고 37℃/5% CO2 인큐베이터 내에서 16시간 배양한 후 ELISA 리더기를 사용하여 560 nm, 650 nm 내 흡광도를 측정하여 세포 증식 능력을 분석하였다. In order to evaluate the cytotoxicity of Bifenthrin, pTr and pLE cells cultured in 96 well were treated with Bifenthrin and cultured for 48 hours. Then, 10 μL of MTT labeling reagent was additionally dispensed and incubated at 37° C. for 4 hours. After dispensing 100 μL of solubilization solution into each well and incubating in an incubator at 37℃/5% CO 2 for 16 hours, cell proliferation ability was analyzed by measuring absorbance at 560 nm and 650 nm using an ELISA reader.
TrypanTrypan blue 염색을 이용한 세포 증식 능력 분석 Analysis of cell proliferation ability using blue staining
비펜트린의 증식 억제 효과를 확인하기 위하여, 비펜트린 (0, 2, 5, 10 μM)을 용량 의존적으로 처리한 배지에서 pTr 및 pLE 세포주를 24시간, 48시간, 72시간 동안 배양하였다. 이후, 0.05% 트립신을 사용하여 세포를 배양 접시에서 떼어낸 후 Trypan blue를 이용하여 염색하였다. 세포계수기 (Hematocytometer)에 20 μL의 염색된 세포를 접종하여 살아있는 세포의 수와 죽은 세포의 수를 카운팅한 뒤, 다음의 방정식을 통해 총 세포 수를 계산하였다. (카운팅한 세포의 수) / 4 × (희석 계수) × (세포 현탁액의 부피). 또한, 세포 생존력은 (살아있는 세포의 수) / (총 세포 수) × 100으로 계산하였으며, 실험은 3번 반복하여 수행하였다. In order to confirm the proliferation inhibitory effect of bifenthrin, pTr and pLE cell lines were cultured for 24 hours, 48 hours, and 72 hours in media treated with bifenthrin (0, 2, 5, 10 μM) in a dose-dependent manner. Thereafter, the cells were removed from the culture dish using 0.05% trypsin and then stained using Trypan blue. After inoculating 20 μL of the stained cells in a hematocytometer and counting the number of live and dead cells, the total number of cells was calculated through the following equation. (number of cells counted) / 4 × (dilution factor) × (volume of cell suspension). In addition, cell viability was calculated as (number of living cells) / (total number of cells) × 100, and the experiment was repeated three times.
Transwell을Transwell 이용한 세포 이동 능력 분석 Cell migration ability analysis using
pTr 세포에 10 μM의 비펜트린 또는 DMSO (대조군)를 처리하여 Transwell inserts (catalog number: 3422, Corning, Corning, NY, USA)에서 14시간 동안 배양하였다. 이후, 메탄올을 이용하여 10분간 세포를 고정시킨 뒤 hematoxylin을 이용하여 상온에서 30분 동안 염색하였다. 염색이 끝난 후, 남은 hematoxylin은 1차수를 이용하여 제거하고 single blade를 이용하여 membrane을 분리하였다. Membrane은 마운팅을 통해 슬라이드에 고정한 뒤, 광학현미경 DM3000 (Leica, Wetzlar, Germany)을 이용해 촬영하였다.pTr cells were treated with 10 μM bifenthrin or DMSO (control) and cultured for 14 hours in Transwell inserts (catalog number: 3422, Corning, Corning, NY, USA). Thereafter, the cells were fixed with methanol for 10 minutes and then stained with hematoxylin for 30 minutes at room temperature. After staining, the remaining hematoxylin was removed using primary water and the membrane was separated using a single blade. Membrane was fixed to the slide through mounting and then photographed using an optical microscope DM3000 (Leica, Wetzlar, Germany).
AnnexinAnnexin V와 with V PropidiumPropidium iodide (PI) 염색을 통한 세포사멸 분석 Apoptosis analysis by iodide (PI) staining
비펜트린에 의한 세포 사멸 효과를 확인하기 위하여 FITC Annexin V 세포 사멸 진단 키트 I (BD Biosciences)을 이용하여 실험을 진행하였다. pTr, pLE 세포의 경우, 6 well 배양 접시에 1 x 105 개의 세포를 배양하고 비펜트린 (0, 2, 5, 10 μM)을 48시간 동안 처리하였다. 그 후, 트립신을 이용하여 세포를 배양 접시에서 떼어낸 뒤 PBS로 워싱하였다. 원심분리하여 얻은 세포를 1x Annexin binding buffer에 현탁배양하여 Annexin V와 PI를 각각 5 μL씩 첨가하고 15분 동안 염색하였다. 형광 신호는 유세포 분석기를 사용하여 측정하였다.In order to confirm the apoptosis effect by bifenthrin, an experiment was performed using FITC Annexin V Cell Death Diagnostic Kit I (BD Biosciences). In the case of pTr and pLE cells, 1 x 10 5 cells were cultured in a 6-well culture dish and treated with bifenthrin (0, 2, 5, 10 μM) for 48 hours. Thereafter, the cells were removed from the culture dish using trypsin and washed with PBS. Cells obtained by centrifugation were cultured in suspension in 1x Annexin binding buffer, and 5 μL each of Annexin V and PI were added and stained for 15 minutes. Fluorescent signals were measured using a flow cytometer.
면역형광법을immunofluorescence method 이용한 세포 증식 cell proliferation using 마커의marker's 발현 분석 Expression analysis
Confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea)에 pTr, pLE 세포를 배양한 뒤, 비펜트린 10 μM을 24시간 동안 처리하였다. 메탄올을 이용하여 세포를 고정한 후, PCNA 항체를 처리하여 4℃에서 16시간 인큐베이션 하였다. 이후 2차 항체로는 goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA)을 antibody dilution buffer에 1:200으로 희석하여 상온에서 1시간 동안 반응시켰다. 0.1% BSA-PBS로 워싱하여 결합하지 않은 2차 항체를 제거해 준 다음 DAPI를 이용하여 핵을 염색했다. 형광 신호는 LSM710(Carl Zeiss, Thornwood, NY, USA) 공초점 현미경을 통해 촬영하였다.After culturing pTr and pLE cells in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea), they were treated with 10 μM of bifenthrin for 24 hours. After fixing the cells with methanol, they were treated with PCNA antibody and incubated at 4°C for 16 hours. Subsequently, as a secondary antibody, goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA) was diluted 1:200 in antibody dilution buffer and reacted at room temperature for 1 hour. After washing with 0.1% BSA-PBS to remove unbound secondary antibodies, the nuclei were stained with DAPI. Fluorescent signals were captured using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.
PI 염색을 통한 세포 주기 진행의 분석Analysis of cell cycle progression via PI staining
세포주기 분석을 위해 propidium iodide 염색을 통해 DNA양을 측정하였다. 비펜트린 (0, 2, 5, 10 μM)을 48시간 동안 처리한 pTr, pLE 세포를 트립신을 이용하여 배양 접시에서 떼어낸 후 에탄올을 이용하여 16시간 동안 고정시켰다. 고정된 세포를 RNase A (Sigma)와 PI (BD Biosciences)로 염색하여 유세포분석을 통해 형광 신호를 측정하였다. 이후 PI 신호 정량을 통해 각 세포 주기의 비율을 분석하였다.For cell cycle analysis, the amount of DNA was measured by propidium iodide staining. The pTr and pLE cells treated with bifenthrin (0, 2, 5, and 10 μM) for 48 hours were detached from the culture dish using trypsin, and then fixed using ethanol for 16 hours. The fixed cells were stained with RNase A (Sigma) and PI (BD Biosciences), and fluorescence signals were measured through flow cytometry. Then, the ratio of each cell cycle was analyzed through PI signal quantification.
DCFHDCFH -DA 염색을 이용한 세포 내 활성산소 분석-Intracellular active oxygen analysis using DA staining
세포 내 활성산소 분석은 DCFH-DA 염색을 통해 실험을 진행하였다. 먼저 100π 배양접시에 배양한 pTr, pLE 세포를 떼어낸 후, DCFH-DA (Sigma)와 함께 30분 동안 함께 배양하여 세포 내로 침투시켰다. 이후 pTr, pLE 세포에 비펜트린 (0, 2, 5, 10 μM)을 1시간 처리하였다. 추가로 활성산소 수준 회복을 검증하기 위해 스캐빈저인 NAC (N-acetyl-1-glutamine)를 병용처리하였다. 최종적으로 DCFH-DA의 형광 신호는 유세포 분석기를 사용하여 측정하였다. Intracellular active oxygen analysis was conducted through DCFH-DA staining. First, pTr and pLE cells cultured in a 100π culture dish were removed, and then incubated with DCFH-DA (Sigma) for 30 minutes to infiltrate the cells. Thereafter, the pTr and pLE cells were treated with Bifenthrin (0, 2, 5, 10 μM) for 1 hour. In addition, to verify the recovery of active oxygen levels, a scavenger, N-acetyl-1-glutamine (NAC), was co-treated. Finally, the fluorescence signal of DCFH-DA was measured using a flow cytometer.
JCJC -1 염색을 이용한 미토콘드리아 Mitochondria using -1 staining 막전위membrane potential 분석 analyze
Mitochondrial staining kit (Sigma)를 사용해 미토콘드리아 막전위를 측정하였다. 비펜트린 (0, 2, 5, 10 μM)을 48시간 동안 처리한 pTr, pLE 세포를 배양 접시에서 떼어낸 후 원심분리하여 세포 pellet을 얻었다. 이후, 세포를 JC-1 staining solution에서 37℃에 20분 동안 배양한 뒤, JC-1 staining buffer를 이용해 워싱하였다. 이후 세포 내 JC-1의 형광 신호는 유세포 분석기를 사용하여 검출하였다. Mitochondrial membrane potential was measured using a Mitochondrial staining kit (Sigma). The pTr and pLE cells treated with bifenthrin (0, 2, 5, and 10 μM) for 48 hours were removed from the culture dish and centrifuged to obtain a cell pellet. Thereafter, the cells were incubated in JC-1 staining solution at 37° C. for 20 minutes, and then washed using JC-1 staining buffer. Thereafter, the fluorescence signal of JC-1 in cells was detected using a flow cytometer.
FluoFluo -4 염색을 이용한 세포질 칼슘 농도 분석Analysis of cytosolic calcium concentration using -4 staining
비펜트린이 세포 내 Ca2 + 농도에 미치는 영향을 확인하기 위하여 먼저 6 well에 배양한 pTr, pLE 세포에 비펜트린 (0, 25, 50, 100 μM)을 용량의존적으로 처리하여 48시간 동안 배양하였다. 이후, 트립신을 이용해 배양 접시에서 세포를 떼어내고 Fluo-4 AM (Invitrogen)과 함께 20분 동안 인큐베이션 하였다. 염색된 세포는 PBS를 이용해 워싱 과정을 거친 뒤 유세포 분석기를 사용하여 형광 신호를 분석하였다.In order to confirm the effect of Bifenthrin on intracellular Ca 2+ concentration, pTr and pLE cells cultured in 6 wells were dose-dependently treated with Bifenthrin (0, 25, 50, 100 μM) and cultured for 48 hours. did Thereafter, cells were detached from the culture dish using trypsin and incubated with Fluo-4 AM (Invitrogen) for 20 minutes. The stained cells were washed with PBS, and fluorescence signals were analyzed using a flow cytometer.
RhodRhod -2 염색을 이용한 미토콘드리아 칼슘 농도 분석Analysis of mitochondrial calcium concentration using -2 staining
미토콘드리아의 칼슘 수준을 검출하기 위해 미토콘드리아를 통과하여 내부의 칼슘 이온과 결합하는 Rhod-2 AM 형광 단백질을 이용한 실험을 진행하였다. 비펜트린을 처리한 pTr, pLE 세포를 Rhod-2 AM을 이용해 염색한 뒤, 결합하지 않고 남은 Rhod-2 AM을 제거하기 위해 HBSS 배지를 이용해 워싱하였다. 염색된 세포를 Ca2 + Mg2 +이 포함되지 않은 HBSS 배지에 10분간 37℃에서 인큐베이션 시킨 뒤, 분석을 진행하였다. Rhod-2 AM의 형광 강도는 유세포 분석기를 이용해 측정하였다. In order to detect the level of mitochondrial calcium, an experiment was conducted using Rhod-2 AM fluorescent protein that passes through mitochondria and binds to calcium ions inside. The bifenthrin-treated pTr and pLE cells were stained with Rhod-2 AM and then washed with HBSS medium to remove unbound Rhod-2 AM. The stained cells were incubated in HBSS medium without Ca 2 + Mg 2+ for 10 minutes at 37° C., and analysis was performed. The fluorescence intensity of Rhod-2 AM was measured using a flow cytometer.
웨스턴블롯을western blot 이용한 단백질 발현 수준 분석 Protein expression level analysis using
비펜트린을 처리한 pTr, pLE 세포로부터 전체 단백질을 추출하여 Bradford protein assay를 통해 단백질 농도를 측정하였다. 이후, 추출한 단백질을 95℃에서 5분 동안 변성하였으며 SDS/PAGE 젤에서 전기영동을 수행하였다. 이를 nitrocellulose membrane으로 transfer 시킨 뒤, 1차 항체와 2차 항체를 차례로 인큐베이션하였다. membrane에 부착된 항체를 chemiluminescenc detection (SuperSignal West Pico, Pierce) 시약을 사용하여 반응시킨 뒤 ChemiDoc EQ system을 통해 단백질 발현을 분석하였다.Total protein was extracted from bifenthrin-treated pTr and pLE cells, and protein concentration was measured by Bradford protein assay. Thereafter, the extracted proteins were denatured at 95° C. for 5 minutes, and electrophoresis was performed on an SDS/PAGE gel. After transferring this to a nitrocellulose membrane, the primary antibody and the secondary antibody were sequentially incubated. Antibodies attached to the membrane were reacted using chemiluminescenc detection (SuperSignal West Pico, Pierce) reagent, and protein expression was analyzed using the ChemiDoc EQ system.
통계분석statistical analysis
본 실험결과는 SAS (statistical analysis system) 통계프로그램을 이용하여 평균과 표준오차를 계산하였고, 일원배치분산분석 (one-way ANOVA)을 실시하였다. P < 0.05 수준에서 유의성 검정을 실시하였다. For the results of this experiment, the mean and standard error were calculated using the statistical analysis system (SAS) statistical program, and one-way ANOVA was performed. Significance tests were performed at the P < 0.05 level.
결과 및 고찰Results and Discussion
비펜트린에to bifenthrin 의한 돼지 Pig by 영양막Trophoblast 세포 및 자궁내막 세포의 증식 억제 효과 Cell and endometrial cell proliferation inhibitory effect
비펜트린이 영양막 세포와 자궁내막 세포의 증식에 미치는 영향을 확인하기 위하여, 비펜트린을 돼지의 영양막세포주(pTr)와 자궁내막세포주(pLE)에 용량의존적으로 (0, 1, 2, 5, 10, 20, 30, 50 μM) 처리한 후, 세포생존력의 변화를 확인하였다. 그 결과, 비펜트린의 농도가 증가함에 따라 pTr, pLE 세포의 증식력은 최대 30%까지 감소하는 것을 확인할 수 있었다 (도 1). 이를 통해 비펜트린이 영양막 세포와 자궁내막 세포의 증식력을 유의적으로 감소시킬 수 있음을 확인하였다. To confirm the effect of bifenthrin on the proliferation of trophoblast and endometrial cells, bifenthrin was dose-dependently (0, 1, 2, 5, 10, 20, 30, 50 μM), changes in cell viability were confirmed. As a result, it was confirmed that the proliferative capacity of pTr and pLE cells decreased by up to 30% as the concentration of bifenthrin increased (FIG. 1). Through this, it was confirmed that bifenthrin can significantly reduce the proliferation of trophoblast and endometrial cells.
비펜트린에to bifenthrin 의한 돼지 Pig by 영양막세포trophoblast cells 및 and 자궁내막세포의endometrial cells 세포 사멸 효과 cell death effect
다음으로 처리 시간에 따른 비펜트린의 효과를 확인하기 위하여 pTr, pLE 세포에 비펜트린 (0, 2, 5, 10 μM)을 처리하고 24, 48, 72시간 뒤 trypan blue 염색을 진행하였다. 그 결과, 대조군에비해 비펜트린 처리 세포에서 시간에 따라 세포 증식 속도가 감소함을 확인하였다 (도 2A). 추가적으로 총 세포 수에서 살아있는 세포의 비율을 분석한 결과, 비펜트린 처리 시간이 길어질수록 농도에 따른 생존률 감소 기울기가 유의적으로 커지는 것을 확인하였다 (도 2B). 또한 Annexin V 실험을 통해 비펜트린의 세포사멸 유도 효과를 확인한 결과, pTr, pLE 세포에서 초기 세포사멸과 후기 세포사멸 모두 유의적으로 증가하는 것을 확인하였다 (도 2C, D). 이러한 결과를 통해 비펜트린은 영양막세포 및 자궁내막세포에서 세포 증식을 저해하고 세포 사멸을 유도하는 효과가 있음을 확인하였다.Next, in order to confirm the effect of bifenthrin according to the treatment time, pTr and pLE cells were treated with bifenthrin (0, 2, 5, and 10 μM), and trypan blue staining was performed 24, 48, and 72 hours later. As a result, it was confirmed that the cell proliferation rate decreased over time in bifenthrin-treated cells compared to the control group (FIG. 2A). Additionally, as a result of analyzing the ratio of viable cells to the total number of cells, it was confirmed that the slope of the decrease in viability according to the concentration significantly increased as the treatment time of Bifenthrin increased (FIG. 2B). In addition, as a result of confirming the apoptosis inducing effect of bifenthrin through Annexin V experiments, it was confirmed that both early and late apoptosis significantly increased in pTr and pLE cells (FIG. 2C, D). Through these results, it was confirmed that bifenthrin has an effect of inhibiting cell proliferation and inducing cell death in trophoblast and endometrial cells.
비펜트린에to bifenthrin 의한 돼지 Pig by 영양막세포trophoblast cells 및 and 자궁내막세포의endometrial cells 세포주기 억제 효과 cell cycle inhibitory effect
다음으로 비펜트린의 세포증식 저해효과를 분자 수준에서 검증하기 위해 세포 증식에 필수 단백질인 PCNA의 발현 변화를 조사한 결과, PCNA 발현을 의미하는 녹색 형광의 세기가 비펜트린의 처리에 따라 감소하는 것으로 나타났 (도 3A, B). 또한, PI 염색을 통해 세포주기를 분석한 결과, 비펜트린 처리가 pTr, pLE 세포에서 sub-G1기 세포 비율을 약 4배 이상 증가시키는 것을 확인할 수 있었다 (도 3C, D). 더불어 세포주기 진행에 필요한 사이클린의 유전자 발현 검사를 통해, 비펜트린의 세포주기 억제가 CCND1과 CCNE1을 감소시킴으로써 유도된다는 것을 확인하였다 (도 3E).Next, to verify the cell proliferation inhibitory effect of Bifenthrin at the molecular level, we investigated changes in the expression of PCNA, an essential protein for cell proliferation. was found (Fig. 3A, B). In addition, as a result of cell cycle analysis through PI staining, it was confirmed that bifenthrin treatment increased the ratio of sub-G1 phase cells by about 4-fold or more in pTr and pLE cells (Fig. 3C, D). In addition, through gene expression tests of cyclins required for cell cycle progression, it was confirmed that bifenthrin's cell cycle inhibition was induced by reducing CCND1 and CCNE1 (FIG. 3E).
비펜트린에to bifenthrin 의한 돼지 Pig by 영양막세포trophoblast cells 및 and 자궁내막세포의endometrial cells 미토콘드리아 항상성 붕괴와 그에 따른 활성산소의 발생 Disruption of mitochondrial homeostasis and consequent generation of reactive oxygen species
비펜트린이 미토콘드리아의 항상성에 미치는 영향을 확인하기 위해 JC-1 분석을 진행하였다. 그 결과, 비펜트린의 처리에 따라 pTr, pLE 세포 내에서 미토콘드리아 탈분극의 지표인 JC-1의 신호가 각각 5.5배, 3.8배 증가하는 것으로 나타났다 (도 4A, B). 미토콘드리아는 활성산소의 주요 발생원이므로, DCFH-DA 염색을 통해 비펜트린 처리에 따른 자궁내막세포 내 활성산소 발생 여부를 확인하였다. 예상대로 비펜트린은 pTr, pLE 세포 내 활성산소를 약 1.5배 증가시켰으며 활성산소 저해제로 알려진 NAC을 함께 처리했을 때 세포 내 활성산소 수준이 회복되는 것을 확인할 수 있었다 (도 4C, D). 추가적으로, 비펜트린에 의해 발생한 활성산소가 실제 세포에 손상을 유도하는지 검증하기 위해 pTr, pLE 세포 내 지질과산화를 확인한 결과, 비펜트린 처리 시 지질 산화의 생성물을 검출하는 녹색 형광 신호가 증가함을 확인할 수 있었다 (도 4E, F). 이러한 결과를 통해, 비펜트린이 돼지 영양막세포 및 자궁내막세포에서 미토콘드리아 탈분극을 유도하여 활성산소 발생을 유발한다는 것을 확인하였다. JC-1 assay was performed to confirm the effect of bifenthrin on mitochondrial homeostasis. As a result, the signal of JC-1, which is an indicator of mitochondrial depolarization, increased 5.5-fold and 3.8-fold, respectively, in pTr and pLE cells according to bifenthrin treatment (FIG. 4A, B). Since mitochondria are a major source of reactive oxygen species, DCFH-DA staining was performed to determine whether reactive oxygen species were generated in endometrial cells following bifenthrin treatment. As expected, Bifenthrin increased intracellular ROS by about 1.5-fold in pTr and pLE, and it was confirmed that intracellular ROS levels were restored when treated together with NAC, known as an ROS inhibitor (FIG. 4C, D). Additionally, as a result of confirming lipid peroxidation in pTr and pLE cells to verify that reactive oxygen species generated by bifenthrin actually induce cell damage, green fluorescence signals detecting products of lipid oxidation increased when bifenthrin was treated. was confirmed (Fig. 4E, F). Through these results, it was confirmed that bifenthrin induces the generation of reactive oxygen species by inducing mitochondrial depolarization in porcine trophoblast and endometrial cells.
영양막세포trophoblast cells 및 and 자궁내막세포에to endometrial cells 작용하는 working 비펜트린의of bifenthrin 소포체 스트레스 유도 및 칼슘 조절 저해 효과 분석 Analysis of endoplasmic reticulum stress induction and calcium regulation inhibitory effect
비펜트린이 세포 내 활성산소를 생성하고 미토콘드리아 기능을 손상시킨다는 점에 착안하여 소포체 스트레스를 유발할 것이라 예상하였다. 이를 검증하기 위해 웨스턴블랏을 통해 소포체 내 스트레스 반응에 관여하는 신호전달 단백질의 발현을 측정하였다. 그 결과, 비펜트린의 농도가 증가함에 따라 대표적인 샤페론 단백질인 GRP78의 발현이 유의적으로 증가하였으며, 번역개시 복합체의 구성 인자인 phsophorylated-eIF2α와 전사 인자 GADD153의 발현이 모두 증가함을 확인할 수 있었다 (도 5A, B). 또한, 소포체 기능의 손상은 세포 내 칼슘 이온 조절에 큰 영향을 미치므로 Rhod2-AM과 Fluo4-AM 염색을 통해 세포 내 칼슘 변화를 분석하였다. 먼저, 유세포분석을 통해 Rhod2-AM의 형광 신호를 분석한 결과, 비펜트린에 의해 pTr, pLE 세포의 미토콘드리아 칼슘이 약 160%, 180% 증가했음을 확인하였다 (도 5C, D). 또한 Fluo4-AM 분석 결과를 통해 세포질 칼슘도 각각 110%, 150% 증가하는 것을 확인할 수 있었다 (도 5E, F). 이러한 결과를 통해, 비펜트린이 소포체 스트레스 반응을 유도하여 세포 내 칼슘 항상성 조절 능력을 저해한다는 것을 확인하였다. Considering that bifenthrin generates intracellular reactive oxygen species and impairs mitochondrial function, it was expected to induce endoplasmic reticulum stress. To verify this, the expression of signaling proteins involved in the stress response in the endoplasmic reticulum was measured by Western blotting. As a result, as the concentration of bifenthrin increased, the expression of GRP78, a representative chaperone protein, increased significantly, and the expression of both phsophorylated-eIF2α, a component of the translation initiation complex, and the transcription factor GADD153 increased. (Fig. 5A, B). In addition, since damage to endoplasmic reticulum function has a great effect on intracellular calcium ion regulation, changes in intracellular calcium were analyzed using Rhod2-AM and Fluo4-AM staining. First, as a result of analyzing the fluorescence signal of Rhod2-AM through flow cytometry, it was confirmed that bifenthrin increased mitochondrial calcium in pTr and pLE cells by about 160% and 180% (Fig. 5C, D). In addition, through the results of Fluo4-AM analysis, it was confirmed that cytoplasmic calcium increased by 110% and 150%, respectively (FIG. 5E, F). Through these results, it was confirmed that bifenthrin inhibits the ability to regulate intracellular calcium homeostasis by inducing the endoplasmic reticulum stress response.
돼지의 pig's 영양막세포trophoblast cells 및 and 자궁내막세포에to endometrial cells 작용하는 working 비펜트린의of bifenthrin 신호전달경로 조절 효과 분석 Signal transduction pathway regulation effect analysis
앞서 확인한 세포 내 스트레스 반응 및 항상성 붕괴가 세포 내 생존 신호전달 경로에 영향을 미칠 것이라는 가정하에 웨스턴블롯 분석을 통해 PI3K 및 MAPK 경로의 단백질 활성화 양상을 조사하였다. 그 결과, pTr 세포에서 비펜트린의 처리 농도에 따라 AKT, p70S6K, S6, ERK1/2, JNK. p38 단백질의 인산화가 모두 유의적으로 증가하는 것으로 나타났다 (도 6A, C). 또한, pLE 세포에서는 MAPK 경로가 증가하는 양상을 보였으며 (도 6B), PI3K 경로의 AKT와 p70S6K의 인산화는 비펜트린 처리에 따라 감소하는 것으로 나타났다 이때, 상위 인자들의 경향성과는 달리 S6 단백질의 인산화는 약간 증가하였다 (도 6D). 이러한 결과를 통해, 비펜트린이 돼지 영양막 세포에서 MAPK 및 PI3K 신호전달 경로를 활성화 시키고, 자궁내막 세포에서는 MAPK 경로를 활성화, PI3K 경로를 억제시킴을 확인하였다.Under the assumption that the above-identified intracellular stress response and homeostasis disruption would affect the intracellular survival signaling pathway, protein activation patterns of the PI3K and MAPK pathways were investigated through Western blot analysis. As a result, AKT, p70S6K, S6, ERK1/2, and JNK were detected in pTr cells according to the treatment concentration of Bifenthrin. Phosphorylation of p38 protein was all significantly increased (Fig. 6A, C). In addition, the MAPK pathway was increased in pLE cells (FIG. 6B), and the phosphorylation of AKT and p70S6K in the PI3K pathway decreased with bifenthrin treatment. Phosphorylation was slightly increased (Fig. 6D). Through these results, it was confirmed that bifenthrin activated the MAPK and PI3K signaling pathways in porcine trophoblast cells, and activated the MAPK pathway and inhibited the PI3K pathway in endometrial cells.
비펜트린에to bifenthrin 의한 돼지의 of pigs by 영양막세포trophoblast cells 및 and 자궁내막세포endometrial cells 스페로이드spheroid 형성 및 성장 저해 효과 formation and growth inhibitory effects
비펜트린이 부착배양이 아닌 3D 배양 세포의 생존능력도 저해하는지 확인하기 위해 spheroid 세포 배양을 진행하였다. 그 결과, 10 μM의 비펜트린은 pTr, pLE 세포의 증식을 억제하여 세포의 spheroid 성장을 감소시켰다 (도 7A). 또한, Image J 프로그램을 이용하여 pTr 및 pLE 세포 스페로이드의 콜로니 수와 총 면적을 분석한 결과, 비펜트린 처리에 따라 콜로니 수는 각각 60%, 73%까지 감소하고, 크기는 75%, 80%까지 감소하는 것을 확인할 수 있었다 (도 7B, C). 이러한 결과를 통해 3D 구조를 형성하고 있는 돼지의 영양막 세포 및 자궁내막 세포에서도 비펜트린이 세포 증식을 현저하게 억제시키는 효과가 있음을 확인하였다. Spheroid cell culture was performed to determine whether bifenthrin also inhibited the viability of 3D cultured cells, not adherent culture. As a result, 10 μM of bifenthrin inhibited the proliferation of pTr and pLE cells and reduced the growth of spheroid cells (Fig. 7A). In addition, as a result of analyzing the colony number and total area of pTr and pLE cell spheroids using the Image J program, the number of colonies decreased by 60% and 73%, respectively, and the size decreased by 75% and 80%, respectively, according to bifenthrin treatment. % was confirmed (Fig. 7B, C). Through these results, it was confirmed that bifenthrin had an effect of significantly inhibiting cell proliferation in porcine trophoblast and endometrial cells forming a 3D structure.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시형태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (8)
상기 자궁내막 세포의 비정상적 증식 관련 질환은 자궁내막증(endometriosis), 자궁내막증식증(endometrial hyperplasia), 자궁내막 폴립(endometrial polyp) 및 자궁내막암(endometrial cancer)으로 이루어진 군에서 선택되는 1종 이상인 것을 특징으로 하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물.According to claim 1,
The disease associated with abnormal proliferation of endometrial cells is at least one selected from the group consisting of endometriosis, endometrial hyperplasia, endometrial polyp, and endometrial cancer. A pharmaceutical composition for preventing or treating abnormal proliferation-related diseases of endometrial cells or trophoblast cells.
상기 영양막 세포의 비정상적 증식 관련 질환은 임신성 영양막 질환(Gestational trophoblastic disease)인 것을 특징으로 하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물.According to claim 1,
A pharmaceutical composition for preventing or treating abnormal proliferation-related diseases of endometrial cells or trophoblastic cells, characterized in that the abnormal proliferation-related disease of trophoblastic cells is gestational trophoblastic disease.
상기 임신성 영양막 질환은 융모막암(choriocarcinoma), 포상기태(hydatidiform mole) 및 융모선종(villous adenoma)로 이루어진 군에서 선택되는 1종 이상인 것을 특징으로 하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물.According to claim 3,
Prevention of diseases associated with abnormal proliferation of endometrial cells or trophoblast cells, characterized in that the gestational trophoblast disease is at least one selected from the group consisting of choriocarcinoma, hydatidiform mole, and villous adenoma. or a pharmaceutical composition for treatment.
상기 비펜트린이 PI3K/AKT 및 MAPK 신호전달기전을 조절하는 것을 특징으로 하는 자궁내막 세포 또는 영양막 세포의 비정상적 증식 관련 질환의 예방 또는 치료용 약학적 조성물.According to claim 1,
A pharmaceutical composition for preventing or treating abnormal proliferation-related diseases of endometrial cells or trophoblast cells, characterized in that the bifenthrin regulates PI3K/AKT and MAPK signaling mechanisms.
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