KR101421325B1 - Composition Comprising Carbon Monoxide or Carbon Monoxide Donor for Inhibition of Bone Loss - Google Patents
Composition Comprising Carbon Monoxide or Carbon Monoxide Donor for Inhibition of Bone Loss Download PDFInfo
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- KR101421325B1 KR101421325B1 KR1020110026216A KR20110026216A KR101421325B1 KR 101421325 B1 KR101421325 B1 KR 101421325B1 KR 1020110026216 A KR1020110026216 A KR 1020110026216A KR 20110026216 A KR20110026216 A KR 20110026216A KR 101421325 B1 KR101421325 B1 KR 101421325B1
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- KR
- South Korea
- Prior art keywords
- carbon monoxide
- present
- bone
- inhibiting
- osteoclast differentiation
- Prior art date
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
본 발명은 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 골 손실 억제용 조성물에 관한 것이다. 본 발명에 따른 일산화탄소 또는 일산화탄소 공급체는 RANKL의 자극에 인해 유발되는 파골세포 분화를 억제하는 효과가 있으며, 이렇게 파골세포 분화를 억제시킴으로써 궁극적으로 파골세포에 의한 골 흡수를 억제하는 효과를 갖게 된다. 더욱 자세하게는, 본 발명에 따른 일산화탄소 또는 일산화탄소 공급체는 RANKL의 자극에 의해 유도된 분자적 신호경로(signal pathway)에서 파골세포 분화를 조절할 수 있는 핵심 전사인자인 NFAT2와 c-fos의 발현 억제 및 NF-κB의 활성화를 억제하는 기전을 통하여 파골세포의 분화를 억제시키는 효과를 갖는다. 또한, 본 발명에 따른 일산화탄소 또는 일산화탄소 공급체는 난소절제술을 시행하여 인위적으로 폐경기를 유도한 마우스 모델에 일산환탄소 공급체(CORM2)를 투입한 경우 골밀도, 뼈의 무기질함량을 증가시키는 효과가 있었다. 따라서 상기와 같은 효과를 갖는 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 본 발명의 조성물은 골 손실로 일어나는 골대사 질환 예방 또는 치료에서 유용하게 사용될 수 있으며, 특히 폐경기 후 골 손실에 따른 골다공증 개선 및 치료하기 위한 용도로 널리 사용할 수 있다.The present invention relates to a composition for inhibiting bone loss containing carbon monoxide or a carbon monoxide supply body as an active ingredient. The carbon monoxide or carbon monoxide supply body according to the present invention has an effect of inhibiting osteoclast differentiation induced by stimulation of RANKL and thus inhibiting osteoclast differentiation and ultimately inhibiting bone resorption by osteoclasts. More particularly, the carbon monoxide or carbon monoxide feed according to the present invention inhibits the expression of NFAT2 and c-fos, the key transcription factors that can regulate osteoclast differentiation in the molecular signal pathway induced by RANKL stimulation, It has an effect of inhibiting osteoclast differentiation through a mechanism of inhibiting the activation of NF-κB. In addition, the carbon monoxide or carbon monoxide supply body according to the present invention has an effect of increasing the bone mineral density and bone mineral content when a monosaccharide carbon supply body (CORM2) is added to a mouse model artificially induced by menopause by ovariectomy . Therefore, the composition of the present invention containing the carbon monoxide or carbon monoxide supply material having the above-mentioned effects as an effective ingredient can be effectively used in the prevention or treatment of bone metabolic diseases caused by bone loss. In particular, the osteoporosis improvement and treatment It can be widely used for the purpose.
Description
본 발명은 폐경기 후 나타나는 골 손실을 효과적으로 개선시킬 수 있는 조성물에 관한 것으로서, 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 골 손실 조성물에 관한 것이다.The present invention relates to a composition capable of effectively improving bone loss after menopause, and relates to a bone loss composition comprising carbon monoxide or a carbon monoxide supply body as an effective ingredient.
뼈(bone)는 인체의 연조직과 체중을 지탱해주고 내부기관을 둘러싸서 내부 장기를 외부의 충격으로부터 보호한다. 또한 근육이나 장기를 구조적으로 지탱할 뿐만 아니라 체내의 칼슘이나 다른 필수 무기질 즉 인이나 마그네슘과 같은 물질을 저장하는 인체의 중요한 부분 중 하나이다. 따라서 성장이 끝난 성인의 뼈는 멈추지 않고 죽는 날까지 오래된 뼈는 제거하고 새로운 뼈로 대체하는 생성과 흡수과정을 매우 역동적, 지속적으로 반복 재생하면서 균형을 유지하게 된다. 이를 뼈의 재형성(bone remodeling)이라고 한다. 이러한 뼈의 재형성은 성장과 스트레스에 의해서 일어나는 뼈의 미세한 손상을 회복시키고 적절히 뼈의 기능을 유지하는데 필수적이다.The bone supports the body's soft tissues and body weight and surrounds the internal organs to protect the internal organs from external impact. It is also an important part of the body that not only supports the muscles or organs but also stores calcium or other essential minerals, such as phosphorus or magnesium, in the body. Thus, adult bones do not stop growing until the day they die, and the old bones are removed and replaced with new bones. This is called bone remodeling. Such bone reshaping is essential to restore fine bone damage caused by growth and stress and to maintain bone function properly.
뼈의 재형성에는 크게 두 종류의 세포가 관여하는 것으로 알려져 있다. 두 세포 중 하나는 뼈를 생성하는 조골세포(osteoblast)이고, 다른 하나는 뼈를 파괴하는 파골세포(osteoclast)이다. 조골세포는 RANKL(receptor activator of NF-κB ligand: 이하 간략하게 ‘RANKL’이라 함)과 이것의 유도 수용체(decoy receptor)인 OPG(osteoprotegerin)를 생성한다. RANKL이 파골 전구세포(osteoclast progenitor cells) 표면에 있는 수용체인 RANK(receptor activator of NF-κB: 이하 간략하게 ‘RANK'라 함)에 결합하면 파골 전구세포가 파골세포로 분화되어 골 흡수(resorption)가 일어난다. 자세하게는 RANKL 자극에 의한 세포내 칼슘 신호 전달로 파골 전구세포에서 파골세포의 분화를 조절하는 핵심 전사인자로 알려져 있는 NFAT(nuclear factor of activated T cells: 이하 간단하게 ‘NFAT'라 함)의 발현이 유도되고, 그 후 세포외 신호조절인산화효소(extracellular signal-regulated kinase: 이하 간단하게 ‘ERK'라 함), NF-κB, p38 MAP kinase 등의 활성화가 NFAT의 자가증폭(autoamplification)을 유발하여 활성이 지속되는 것이다. It is known that two kinds of cells are involved in bone remodeling. One of the cells is osteoblast that produces bone, and the other is osteoclast that destroys bone. Osteoblasts produce RANKL (receptor activator of NF-κB ligand) and OPG (osteoprotegerin), its decoy receptor. When RANKL binds to RANK (receptor activator of NF-κB), which is a receptor on the surface of osteoclast progenitor cells, osteoclast precursor cells are differentiated into osteoclasts, . In detail, the expression of nuclear factor-activated T cells (NFAT), which is known as a key transcription factor that regulates osteoclast differentiation in osteoclast precursor cells by intracellular calcium signaling by RANKL stimulation, Activation of extracellular signal-regulated kinase (ERK), NF-κB, and p38 MAP kinase induces autoamplification of NFAT. This is what will last.
따라서 오래된 뼈의 흡수 또는 파괴는 상기와 같은 기작을 통해 파골세포에 의해 이루어지며, 이는 뼈에 구멍을 내어 적은 양의 칼슘이 혈류로 방출되어 신체기능을 유지하는데 사용된다(William J. et al., NATURE., 423, pp.337342, 2033). 파골세포는 실제적인 뼈의 흡수작용을 할 때는 다핵세포로 융합된 후 그 구조가 변하여 주름진 테두리를 형성하며, 골 기질 표면에 흡착하여 산성화 환경에서 카텝신(cathepsin) K 나 H+-ATPase 등 많은 효소의 작용으로 뼈를 흡수한다. 이에 반해, 조골세포는 교원질로 구멍을 채우고 칼슘과 인의 침적물(hydroxyapatite)을 덮어서 단단한 새로운 뼈를 만들어 골격을 재건하게 된다.Thus, the absorption or destruction of old bone is carried out by the osteoclast through the above mechanism, which is used to keep the body function by piercing the bone and releasing a small amount of calcium into the bloodstream (William J. et al. , NATURE., 423, pp. 3737342, 2033). When osteoclasts are actually absorbed by bone, they are fused to polynuclear cells, and their structures are changed to form a corrugated border. They are adsorbed on the surface of bone matrix, and in the acidified environment, many enzymes such as cathepsin K and H + -ATPase To absorb the bone. In contrast, osteoblasts fill holes with collagen and cover calcium and phosphorus deposits (hydroxyapatite), creating a firm new bone and rebuilding the skeleton.
성인의 경우 매년 골격의 약 10-30%가 이런 과정을 통하여 재형성되며, 파골속도와 조골 속도가 동일해야만 전과 같은 골밀도를 유지할 수 있다. 정상 성인에서는 골흡수 양과 골형성 양 사이에는 항상 균형이 유지되고 있다. 이와 같이 중요한 뼈에 균형이 깨질 경우 많은 질병이 야기될 수 있는데, 이러한 질환을 골대사 질환이라 한다.In adults, about 10-30% of the bone is reformed every year through this process, and bone density can be maintained as long as the osteopenic rate and osteolysis rate are the same. In normal adults, there is always a balance between the amount of bone resorption and the amount of bone formation. When such an important bone is broken, many diseases can be caused. This disease is called bone disease.
골대사 질환 중 고령사회에서 큰 문제로 대두되고 있는 골다공증은 골의 화학적 조성에는 큰 변화 없이 단위 용적내의 골량(bone mass)이 감소하여 경미한 충격에도 쉽게 골절을 일으킬 수 있는 질환이다. 특히 폐경 후 여성들의 경우 에스트로겐의 분비부족으로 인하여 가장 그 발생빈도가 높게 나타난다고 알려져 있다.Osteoporosis, a major problem in the aging society, is a disease in which the bone mass in the unit volume is decreased without significant change in the bone composition and can easily cause fracture even in the case of a slight impact. In particular, postmenopausal women are known to have the highest incidence of estrogen secretion.
일반적으로 여성이 폐경기에 접어들면 에스트로겐을 생성하지 못하게 되는데, 에스트로겐의 분비 감소는 여성의 비뇨생식계통 뿐만 아니라 신체전반에 걸쳐 큰 영향을 미치게 되며, 배란이 중단되어 월경이 사라지는 것을 폐경이라 한다. 또한, 40세 이전에 발생하는 미숙 폐경은 원인 미확인의 난소 부전으로 통칭되며, 이는 다량의 장기간에 걸친 흡연이나, 고해발 고도에서의 거주, 영양 불량 등과 관련이 있는 것으로 믿어지고 있다, 한편, 인위적 폐경은 난소절제술, 화학요법, 골반에 대한 방사선 조사, 또는 난소에 대한 혈액 공급을 저하시키는 임의의 과정으로부터 초래될 수 있다. 이렇게 폐경이 일어나게 되면, 여러 가지 폐경기 증상이 나타나게 되는데 그 중에 하나가 골 손실에 의한 골다공증인 것이다.In general, when a woman enters the menopause, she will not be able to produce estrogen. The decrease in secretion of estrogen is a major influence not only on the female genitourinary system but also on the whole body, and the termination of ovulation and menstrual cessation is called menopause. In addition, premature menopause occurring before age 40 is commonly referred to as ovarian failure of undetermined cause, which is believed to be associated with large amounts of long-term smoking, residence at elevated altitudes, malnutrition, Menopause may result from ovariectomies, chemotherapy, radiation to the pelvis, or any procedure that reduces blood supply to the ovaries. When menopause occurs, various menopausal symptoms appear, one of which is osteoporosis due to bone loss.
상기와 같이 폐경에 의한 에스트로겐 농도의 급속한 감소는, B-임파구(B-lymphocyte)가 다량 생성되어 골수(bone marrow)에 B 세포 전구체(pre-B cell)가 축적을 야기하고, 이로 인해 IL-6의 양이 증가하여 파골세포의 활성을 증가시키므로 결국 골량이 감소하게 되어 골다공증이 나타나게 된다.As described above, the rapid decrease in estrogen concentration due to menopause causes the accumulation of B-cell precursors (pre-B cells) in the bone marrow due to the generation of a large amount of B-lymphocytes, 6 increases the activity of osteoclasts, resulting in decreased bone mass, resulting in osteoporosis.
한편, 이러한 폐경기 증후군의 치료를 위해 가장 널리 사용되고 있는 방법으로 에스트로겐 대체 요법(estrogen replacement therapy)이 사용되고 있으나, 에스트로겐 요법의 경우 유방암과 같은 특정 암의 발생률이 증가되는 부작용이 있고, 또한 특정 자궁암 발생과도 관련이 있다는 내용이 보고되고 있다. 따라서 이러한 부작용을 감소시키기 위해 프로제스틴(progestin)을 이용하는 치료가 사용되고 있으나, 에스트로겐-프로게스틴 투약과 같은 치료를 받은 폐경 후 여성들은 자주 불쾌한 자궁내의 출혈을 경험하고 있어 부작용 없이 효과적으로 폐경기 증상을 치료할 수 있는 새로운 치료제의 개발이 시급한 실정이다.Estrogen replacement therapy is the most widely used method for the treatment of postmenopausal syndrome. However, estrogen therapy has a side effect of increasing the incidence of certain cancers such as breast cancer, There is also a report on the relationship. Therefore, although progestin-based therapy has been used to reduce these side effects, postmenopausal women who have received treatments such as estrogen-progestin medication often experience unpleasant uterine bleeding, and are therefore able to effectively treat postmenopausal symptoms without side effects. The development of therapeutic agents is urgent.
이에 본 발명자들은 부작용 없이 폐경기로 인해 발생하는 증상을 효과적으로 억제할 수 있는 연구를 진행하던 중, 일산화탄소가 폐경기 후 발생하는 골 손실 증가를 억제할 수 있다는 사실을 확인함으로써 본 발명을 완성하였다.Accordingly, the inventors of the present invention have completed the present invention by confirming that carbon monoxide can inhibit an increase in bone loss occurring after menopause while conducting research to effectively inhibit symptoms caused by menopause without side effects.
따라서 본 발명의 목적은 파골세포의 분화 억제효과가 우수한 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 골 손실 억제용 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a composition for inhibiting bone loss, which contains carbon monoxide or a carbon monoxide supply body having an excellent osteoclast differentiation inhibitory effect as an active ingredient.
또한, 본 발명의 다른 목적은 파골세포의 분화 억제효과가 우수한 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 골 손실 억제용 건강기능식품을 제공하는 것이다. Another object of the present invention is to provide a health functional food for bone loss suppression containing carbon monoxide or a carbon monoxide supply body having an excellent osteoclast differentiation inhibitory effect as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 골 손실 억제용 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a composition for inhibiting bone loss containing carbon monoxide or a carbon monoxide supply body as an active ingredient.
본 발명의 일실시예에 있어서, 상기 일산화탄소 공급체는 CORM2(CO releasing molecular 2)일 수 있다.In one embodiment of the present invention, the carbon monoxide supplier may be CORM2 (CO releasing molecular 2).
본 발명의 일실시예에 있어서, 상기 일산화탄소 또는 일산화탄소 공급체는 폐경기로 인해 유발되는 골 손실을 감소시킬 수 있다.In one embodiment of the present invention, the carbon monoxide or carbon monoxide supply body may reduce bone loss caused by menopause.
본 발명의 일실시예에 있어서, 상기 일산화탄소 또는 일산화탄소 공급체는 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT2, c-fos의 발현 억제 및 NF-κB의 활성화 억제 기전을 통하여 골 손실을 감소시킬 수 있다.In one embodiment of the present invention, the carbon monoxide or carbon monoxide supplier reduces bone loss through inhibition of the expression of NFAT2, c-fos, and NF-κB, which are key transcription factors controlling osteoclast differentiation .
또한, 본 발명은 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 골 손실 억제를 위한 건강기능식품을 제공한다.The present invention also provides a health functional food for inhibiting bone loss containing carbon monoxide or a carbon monoxide supply body as an active ingredient.
본 발명의 일실시예에 있어서, 상기 일산화탄소 공급체는 CORM2(CO releasing molecular 2)일 수 있다.In one embodiment of the present invention, the carbon monoxide supplier may be CORM2 (CO releasing molecular 2).
본 발명의 일실시예에 있어서, 상기 일산화탄소 또는 일산화탄소 공급체는 폐경기로 인해 유발되는 골 손실을 감소시킬 수 있다.In one embodiment of the present invention, the carbon monoxide or carbon monoxide supply body may reduce bone loss caused by menopause.
본 발명에 따른 일산화탄소 또는 일산화탄소 공급체는 RANKL의 자극에 인해 유발되는 파골세포 분화를 억제하는 효과가 있으며, 이렇게 파골세포 분화를 억제시킴으로써 궁극적으로 파골세포에 의한 골 흡수를 억제하는 효과를 갖게 된다. The carbon monoxide or carbon monoxide supply body according to the present invention has an effect of inhibiting osteoclast differentiation induced by stimulation of RANKL and thus inhibiting osteoclast differentiation and ultimately inhibiting bone resorption by osteoclasts.
더욱 자세하게는, 본 발명에 따른 일산화탄소 또는 일산화탄소 공급체는 RANKL의 자극에 의해 유도된 분자적 신호경로(signal pathway)에서 파골세포 분화를 조절할 수 있는 핵심 전사인자인 NFAT2, c-fos의 발현 억제 및 NF-κB의 활성화를 억제하는 기전을 통하여 파골세포의 분화를 억제시키는 효과를 갖는다.More specifically, the carbon monoxide or carbon monoxide feed according to the present invention inhibits the expression of NFAT2, c-fos, which are key transcription factors that can regulate osteoclast differentiation in the molecular signal pathway induced by RANKL stimulation, It has an effect of inhibiting osteoclast differentiation through a mechanism of inhibiting the activation of NF-κB.
또한, 본 발명에 따른 일산화탄소 또는 일산화탄소 공급체는 난소절제술을 시행하여 인위적으로 폐경기를 유도한 마우스 모델에 일산환탄소 공급체(CORM2)를 투입한 경우 골밀도, 뼈의 무기질함량을 증가시키는 효과가 있다.In addition, the carbon monoxide or carbon monoxide supply body according to the present invention has an effect of increasing bone mineral density and bone mineral content when a monosaccharide carbon supply body (CORM2) is added to a mouse model artificially induced by menopause by ovariectomy .
따라서 상기와 같은 효과를 갖는 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 본 발명의 조성물은 골 손실로 일어나는 골대사 질환 예방 또는 치료에서 유용하게 사용될 수 있으며, 특히 폐경기 후 골 손실에 따른 골다공증 개선 및 치료하기 위한 용도로 널리 사용할 수 있다.Therefore, the composition of the present invention containing the carbon monoxide or carbon monoxide supply material having the above-mentioned effects as an effective ingredient can be effectively used in the prevention or treatment of bone metabolic diseases caused by bone loss. In particular, the osteoporosis improvement and treatment It can be widely used for the purpose.
도 1은 본 발명의 일산화탄소 공급체(CORM2)의 처리에 따른 파골세포 분화의 억제정도를 TRAP+ 세포의 개수를 측정하여 나타낸 그래프이다(* : P<0.05, ** : P<0.01, *** : P<0.001, n=3)
도 2는 본 발명의 일산화탄소 공급체(CORM2)의 처리에 따른 파골세포 특이적 마커 유전자(TRACP 및 칼시토닌 수용체)의 발현량을 quantitative PCR로 측정하여 나타낸 그래프이다.
도 3은 본 발명의 일산화탄소 공급체(CORM2)의 시간대별 처리에 따른 파골세포 분화의 억제정도를 나타낸 그래프이다(0-4D : 0일 투입후 96 h 지속, 0-2D : 0일 투입후 48 h에 제거, 3-4D: 49h 투입후 96 h 지속).
도 4는 본 발명의 일산화탄소 공급체(CORM2)의 처리에 따른 파골세포 분화 조절 핵심전사인자(c-fos 및 NEAF2)의 발현량을 quantitative PCR로 측정하여 나타낸 그래프이다.
도 5는 본 발명의 일산화탄소 공급체(CORM2)의 처리에 따른 파골세포 분화 조절 핵심전사인자인 NF-κB의 활성에 미치는 영향을 EMSA 방법을 이용하여 측정한 것이다(lane 1: RANKL 무첨가군, lane 2: RANKL 첨가군, lane 3: RANKL+CORM2 20μM 첨가군, lane 4: RANKL+CORM2 50μM 첨가군, lane 5: mutant competitor probe 사용군).
도 6은 본 발명의 일산화탄소 공급체(CORM2)의 처리에 따른 NFAT2 활성을 가시적으로 확인하기 위하여 면역형광 염색법을 이용하여 다핵 성숙 파골세포로의 핵 이동을 나타낸 이미지이다.
도 7은 난소절제술을 시행한 마우스 모델에 본 발명의 일산화탄소 공급체(CORM2)를 처리한 후 골량의 변화를 확인하기 위하여 대퇴골을 micro CT를 이용하여 단층 촬영한 사진이다.
도 8은 난소절제술을 시행한 마우스 모델에 본 발명의 일산화탄소 공급체(CORM2)를 처리한 후 대퇴부의 해면골의 골밀도(BMD : Bone Mineral Density)를 수치로 나타낸 그래프이다.
도 9는 난소절제술을 시행한 마우스 모델에 본 발명의 일산화탄소 공급체(CORM2)의 처리한 후 뼈의 무기질함량(BMC : bone mineral content)을 수치로 나타낸 그래프이다.
도 10은 난소절제술을 시행한 마우스 모델에 본 발명의 일산화탄소 공급체(CORM2)를 처리한 후 전체 조직 볼륨의 퍼센트로써 표현된 해면골 부피(BV/TV %)를 나타낸 그래프이다.FIG. 1 is a graph showing the degree of inhibition of osteoclast differentiation according to the treatment with the carbon monoxide feeder (CORM2) of the present invention by measuring the number of TRAP + cells (*: P <0.05, **: P <0.01, *** : P < 0.001, n = 3)
FIG. 2 is a graph showing quantitative PCR of the expression levels of osteoclast-specific marker genes (TRACP and calcitonin receptor) according to the treatment of the carbon monoxide feeder (CORM2) of the present invention.
FIG. 3 is a graph showing the degree of inhibition of osteoclast differentiation according to the time-series treatment of the carbon monoxide feeder (CORM2) of the present invention (0-4D: duration of 96 h after 0 day, 0-2D: h, 3-4D: 49 h, then 96 h continuous).
FIG. 4 is a graph showing quantitative PCR of expression levels of osteoclast differentiation-regulating key transcription factors (c-fos and NEAF2) according to the treatment of the carbon monoxide feeder (CORM2) of the present invention.
FIG. 5 shows the effect of the carbon monoxide supplier (CORM2) of the present invention on the activity of NF-κB, which is a key transcription factor for osteoclast differentiation, by the EMSA method (lane 1: no RANKL group, lane 2: RANKL addition, lane 3: 20 μM addition of RANKL + CORM2, lane 4: 50 μM addition of RANKL + CORM2, lane 5: mutant competitor probe group).
FIG. 6 is an image showing nuclear migration to polynuclear mature osteoclast using immunofluorescence staining method to visually confirm NFAT2 activity according to the treatment with the carbon monoxide supply body (CORM2) of the present invention.
FIG. 7 is a photograph of a femur using a micro CT to examine changes in bone mass after treating the carbon monoxide feeder (CORM2) of the present invention with a mouse model subjected to ovariectomy.
8 is a graph showing numerical values of bone mineral density (BMD) of cancellous bone of the thigh after treating the carbon monoxide feeder (CORM2) of the present invention with a mouse model subjected to ovariectomy.
FIG. 9 is a graph showing numerical values of bone mineral content (BMC) after treatment with the carbon monoxide feeder (CORM2) of the present invention in a mouse model subjected to ovariectomy.
FIG. 10 is a graph showing cancellous bone volume (BV / TV%) expressed as a percentage of the total tissue volume after treating the carbon monoxide supplier (CORM2) of the present invention with a mouse model subjected to ovariectomy.
본 발명은 폐경기 후 나타나는 골 손실 증가를 효과적으로 개선시킬 수 있는 신규한 치료제에 관한 것으로서, 보다 구체적으로 본 발명은 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 골 손실 억제용 조성물을 제공함에 그 특징이 있다.The present invention relates to a novel therapeutic agent capable of effectively improving the bone loss appearing after menopause. More specifically, the present invention provides a composition for inhibiting bone loss containing carbon monoxide or a carbon monoxide supply body as an active ingredient. have.
일반적으로 폐경기는 여성에게 있어 월경출혈의 최종 현상으로서 정의된다. 그러나 이 용어는 생식기까지와 월경출혈의 최종 현상을 경과하는 사이의 이행 기간을 포함하여 여성 갱년기의 시기로 언급되는데 통상 사용된다. 또한 이 시기는 폐경기 근처 또는 갱년기로서도 언급되기도 한다. 이러한 시기 동안에는 각종 내분비, 체세포 및 생리적 변화 및 난소 기능의 손실이 점차적으로 진행된다. In general, menopause is defined as the final event of menstrual bleeding in women. However, this term is commonly used to refer to the period of female menopause, including the period of transition between reproductive and late menstrual bleeding. It is also referred to as menopause or as a menopause. During these periods, various endocrine, somatic and physiological changes and loss of ovarian function progress gradually.
또한, 폐경기 근처에서 쇠퇴하는 에스트로겐 수준과 관련된 증상으로는 전신열감 및 발한, 위축질염, 두통, 현기증, 관절통, 불면증, 무감정, 권태, 근육약화, 심계항진 및 분위기 변화, 울병, 기억 및 집중력 저하, 이상감응 및 성적 기능과 관련된 문제와 같은 심리적 증상이 포함되며, 특히 에스트로겐 농도의 급속한 감소는 파골세포의 활성을 증가시켜 골 흡수(골 손실)가 증가하게 되며, 종국에는 골다공증과 같은 골대사 질환을 초래하게 된다.In addition, symptoms associated with estrogen levels declining near menopausal symptoms include general hot sensation and sweating, atrophic vaginitis, headache, dizziness, arthralgia, insomnia, no emotions, boredom, muscle weakness, palpitations and atmospheric changes, And a psychological symptom such as a problem related to abnormal sensation and sexual function. Particularly, a rapid decrease in estrogen concentration increases osteoclast activity, thereby increasing bone resorption (bone loss) and eventually causing bone metabolic diseases such as osteoporosis .
한편, 일산화탄소(CO)는 생물에서 독성 및 치사율을 갖는 기체 분자로 알려져 있다(Haldane, Biochem . J. 21: 1068-1075; 및 Chance 등, 1970, Ann . NY Acad Sci. 174: 193-204). 그러나 최근 보고되고 있는 자료들에 의하면, 일산화탄소가 세포내 및 생물학적 과정에서 여러 반응에 조절 인자로서 작용하며, 특히 뉴런 전송을 매개하고(Verma 등, supra 및 Xhuo 등, Science 260: 1946-1950), 혈관운동 긴장을 조절하는 등(Morita & Kourembanas, 1995, J. Clin. Invest. 96: 2676-2682.; Morita 등, 1995 Proc. Natl. Acad. Sci. USA 92: -1479; 및 Goda 등, 1998, J. Clin. Inv. 101: 604-12)의 역할을 담당하는 것으로 밝혀지고 있다. 따라서 이러한 일산화탄소에 대한 생체 내에서의 생리학적 활성에 대한 연구가 현재 활발히 진행되고 있는데, 본 발명에서는 이러한 일산화탄소가 우수한 파골세포의 분화 억제활성을 가지고 있다는 사실을 확인함으로써, 파골세포의 분화에 의해 유발될 수 있는 골 손실 억제에 유용하다는 사실을 최초로 규명하였다.On the other hand, carbon monoxide (CO) is known as a gas molecule having toxicity and mortality in organisms (Haldane, Biochem J. 21:. . 1068-1075; and the like Chance, 1970, Ann NY Acad Sci. 174: 193-204). However, recently reported data indicate that carbon monoxide acts as a regulator of many responses in intracellular and biological processes, particularly mediating neuronal transmission (Verma et al., Supra and Xhuo et al., Science 260: 1946-1950) (Morita & Kourembanas, 1995, J. Clin. Invest. 96: 2676-2682 . ; Morita et al. , 1995 Proc Natl Acad Sci USA 92: , J. Clin. Inv. 101: 604-12). Therefore, studies on the physiological activity in vivo against carbon monoxide have been actively conducted. In the present invention, by confirming that carbon monoxide has an excellent osteoclast differentiation inhibiting activity, it is possible to induce osteoclast differentiation Which is useful for inhibiting bone loss.
보다 구체적으로, 본 발명에 따른 일산화탄소 또는 일산화탄소 공급체는 RANKL의 자극에 의해 유도된 분자적 신호경로(signal pathway)에서 파골세포 분화를 조절할 수 있는 핵심 전사인자인 NFAT2, c-fos의 발현 억제 및 NF-κB의 활성화를 억제하는 기전을 통하여 파골세포의 분화를 효과적으로 억제할 수 있는 특징이 있다.More specifically, the carbon monoxide or carbon monoxide feed according to the present invention inhibits the expression of NFAT2, c-fos, which are key transcription factors capable of regulating osteoclast differentiation in the molecular signal pathway induced by RANKL stimulation, It is characterized by being capable of effectively inhibiting the differentiation of osteoclasts through a mechanism of inhibiting the activation of NF-κB.
하기 실시예<1-3>에서는 파골세포 분화인자인 RANKL를 마우스 골수세포에 처리하여 파골세포 분화를 유도하는 과정에서, 본 발명의 일산화탄소 공급체(CORM2)를 처리한 경우 파골세포 분화가 억제되는 것으로 나타났다. 따라서 이러한 결과를 통해 본 발명자들은 일산화탄소가 파골세포 분화를 억제하는 효과가 있음을 알 수 있었다.In the following Examples 1-3, RANKL, an osteoclast differentiation factor, is treated on mouse bone marrow cells to induce osteoclast differentiation. When osteoclast differentiation is inhibited when the carbon monoxide supply body (CORM2) of the present invention is treated, Respectively. Therefore, the inventors of the present invention have found that carbon monoxide inhibits osteoclast differentiation.
또한, 하기 실시예<2-1>에서는 파골세포 분화인자인 RANKL를 마우스 골수세포에 처리하여 파골세포 분화를 유도하는 과정에서, 일산화탄소가 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT2 및 c-fos의 발현을 억제할 수 있는지 실험하였는데, 그 결과 본 발명의 일산화탄소 공급체(CORM2)를 처리한 처리군에서 NFAT2 및 c-fos의 발현량이 확실히 감소하는 것으로 나타났다. 따라서 이러한 결과를 통해 본 발명자들은 일산화탄소가 NFAT2 및 c-fos의 발현을 억제하는 기작을 가지며, 이러한 기작으로 파골세포 분화가 억제되는 것을 유추할 수 있었다.In the following Example < 2-1 >, RANKL, which is an osteoclast differentiation factor, was treated with mouse bone marrow cells to induce osteoclast differentiation, NFAT2, which is a key transcription factor regulating osteoclast differentiation, fos expression. As a result, the expression levels of NFAT2 and c-fos were clearly decreased in the treatment group treated with the carbon monoxide feeder (CORM2) of the present invention. Therefore, the inventors of the present invention have been able to deduce that carbon monoxide inhibits the expression of NFAT2 and c-fos, and that this mechanism inhibits osteoclast differentiation.
또한 하기 실시예<2-2>에서는 파골세포 분화인자인 RANKL를 마우스 골수세포에 처리하여 파골세포 분화를 유도하는 과정에서, 일산화탄소가 파골세포의 분화를 조절하는 핵심 전사인자인 NF-κB의 활성화를 억제할 수 있는지 실험하였는데, 그 결과 본 발명의 일산화탄소 공급체(CORM2)를 처리한 처리군에서 NF-κB의 활성이 감소하는 것으로 나타났다. 따라서 이러한 결과를 통해 본 발명자들은 일산화탄소가 NF-κB의 활성화를 억제하는 기작을 가지며, 이러한 기작으로 파골세포 분화가 억제되는 것을 유추할 수 있었다.In the following Example <2-2>, in the process of inducing osteoclast differentiation by treating osteoclast differentiation factor RANKL with mouse bone marrow cells, activation of NF-κB, a key transcription factor that regulates osteoclast differentiation, . As a result, the activity of NF-kB decreased in the treatment group treated with the carbon monoxide feeder (CORM2) of the present invention. Accordingly, the inventors of the present invention have been able to infer that carbon monoxide inhibits the activation of NF-κB, and that this mechanism inhibits osteoclast differentiation.
뿐만 아니라, 하기 실시예 3에서는 난소절제술을 시행하여 인위적으로 폐경기를 유도한 마우스 모델에 일산환탄소 공급체(CORM2)를 투입한 경우 골밀도, 뼈의 무기질함량 및 해면골 부피를 증가시키는 것으로 나타났으며, 이를 통해 in vivo 상에서도 일산화탄소가 골손실 감소를 효과적으로 억제할 수 있음을 증명하였다.In addition, in Example 3 below, when a monoclonal carbon source (CORM2) was injected into a mouse model artificially induced by menopause by ovariectomy, bone mineral density, bone mineral content and cancellous bone volume were increased , Demonstrating that carbon monoxide can effectively inhibit bone loss reduction in vivo.
따라서 상기와 같은 특징을 갖는 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 본 발명의 조성물은 파골세포의 분화 억제를 통하여 골 손실로 인해 발생되는 골대사 질환 예방 또는 치료에 유용하게 사용될 수 있다.Therefore, the composition of the present invention containing carbon monoxide or carbon monoxide supply material having the above characteristics as an active ingredient can be effectively used for prevention or treatment of bone metabolic diseases caused by bone loss through inhibition of osteoclast differentiation.
본 발명의 조성물로 예방 또는 치료할 수 있는 골대사 질환으로는 골다공증, 파제트 골질환, 구루병, 골연화증, 퇴행성 골질환등과 같은 질환일 수 있으나, 가장 바람직하게는 폐경기 후 골 손실에 따른 골다공증일 수 있다.The bone metabolic diseases that can be prevented or treated by the composition of the present invention may be diseases such as osteoporosis, Paget's bone disease, rickets, osteomalacia, degenerative bone disease and the like, but most preferably osteoporosis due to bone loss after menopause .
그러므로 본 발명은 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 골 손실 억제용 조성물을 제공할 수 있다.Therefore, the present invention can provide a bone loss inhibiting composition comprising carbon monoxide or a carbon monoxide supply body as an effective ingredient.
본 발명에서 상기 “일산화탄소(CO)"는 액체 형태로 압출된 또는 수용액에 용해된 기체상인 분자 일산화탄소로 정의할 수 있으며, “일산화탄소를 함유하는 조성물”은 이를 필요로 하는 개체에 처리 또는 투여되어질 수 있는 일산화탄소를 함유하는 기체 또는 액체 조성물을 말한다.In the present invention, the term " carbon monoxide (CO) "may be defined as molecular carbon monoxide, which is a gas phase which is extruded in a liquid form or dissolved in an aqueous solution, and a composition containing carbon monoxide may be treated or administered Refers to a gas or liquid composition containing carbon monoxide.
또한, 본 발명의 조성물에 포함되는 유효성분은 일산화탄소 이외에도 일산화탄소 공급체를 포함한다.In addition, the active ingredient contained in the composition of the present invention includes a carbon monoxide supply body in addition to carbon monoxide.
본 발명에서 사용할 수 있는 상기 일산화탄소 공급체는 이에 제한되지는 않으나, RuCO(Ruthenium Carbon Monoxide), CORM1(Mn2CO10 :manganese decacarbonyl), CORM2([Ru(CO)3Cl2]2), CORM3(Ru(CO)(3)Cl(glycinate)) 및 CORM-A1(sodium boranocarbonate)으로 이루어진 군으로부터 선택된 1종일 수 있으며, 바람직하게는 CORM2([Ru(CO)3Cl2]2)일 수 있다.The carbon monoxide feed material for use with the present invention is, RuCO (Ruthenium Carbon Monoxide), CORM1 but are not limited to, (Mn 2 CO 10: manganese decacarbonyl ), CORM2 ([Ru (CO) 3 Cl 2] 2), CORM3 It can be (Ru (CO) (3) Cl (glycinate)) and CORM-A1 can be a full selected from the group consisting of (sodium boranocarbonate), preferably CORM2 ([Ru (CO) 3 Cl 2] 2) .
본 발명에 따른 조성물은 약제학적 조성물이나 식품 조성물로 사용될 수 있다.The composition according to the present invention can be used as a pharmaceutical composition or a food composition.
본 발명의 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 약제학적 조성물은 상기 유효성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등을 사용할 수 있다.The pharmaceutical composition comprising the carbon monoxide or carbon monoxide supply material of the present invention as an active ingredient may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above active ingredients. Examples of the adjuvants include excipients, A sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.
상기 약제학적 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다. The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.
상기 약제학적 조성물의 제제 형태는 과립제, 산제, 정제, 피복정, 캡슐제, 좌제, 액제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 등이 될 수 있다. 예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효 성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약제학적으로 허용 가능한 불활성 담체와 결합될 수 있다. 또한, 원하거나 필요한 경우, 적합한 결합제, 윤활제, 붕해제 및 발색제 또한 혼합물로 포함될 수 있다. 적합한 결합제는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연 당, 옥수수 감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성 검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐 아세테이트, 소듐 클로라이드 등을 포함한다. 붕해제는 이에 제한되는 것은 아니나, 녹말, 메틸 셀룰로스, 아가, 벤토니트, 잔탄 검 등을 포함한다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 해당분야의 적절한 방법으로 Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화 할 수 있다. The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.
본 발명의 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 식품 조성물은 상기 약제학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.The food composition containing the carbon monoxide or carbon monoxide supply material of the present invention as an active ingredient can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .
본 발명의 식품 조성물은 유효성분인 일산화탄소 또는 일산화탄소 공급체를 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 향미제는 천연 향미제 (타우마틴), 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. The food composition of the present invention may contain, as an additional component, various flavors or natural carbohydrates such as carbon monoxide or a carbon monoxide supplier as an active ingredient, as well as conventional food compositions. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).
또한 상기 식품 조성물은 유효성분인 일산화탄소 또는 일산화탄소 공급체 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. In addition to the carbon monoxide or carbon monoxide supplier, the food composition may further contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and intermediates such as cheese and chocolate, And carbonates used in alginic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
본 발명은 또한 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 함유하는 골 손실 억제를 위한 건강기능식품을 제공한다.The present invention also provides a health functional food for inhibiting bone loss containing carbon monoxide or a carbon monoxide supply body as an active ingredient.
본 발명의 건강기능식품은 골강화 촉진을 목적으로, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings for the purpose of promoting bone strengthening.
본 발명에서 “건강기능식품”이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.
본 발명의 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.
상기 “식품 첨가물 공전”에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다.Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.
예를 들어, 정제 형태의 건강기능식품은 본 발명의 유효성분인 일산화탄소 또는 일산화탄소 공급체를 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수도 있다.For example, the health functional food in the form of tablets may be prepared by granulating a mixture of carbon monoxide or carbon monoxide feed, which is an effective ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives in a conventional manner, Or the mixture can be directly compression-molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.
캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분인 일산화탄소 또는 일산화탄소 공급체를 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 일산화탄소 또는 일산화탄소 공급체를 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.The hard capsule of the capsule type health functional food can be prepared by filling a normal hard capsule with a mixture of carbon monoxide or a carbon monoxide supplier as an active ingredient of the present invention with an additive such as an excipient and the soft capsule is supplied with carbon monoxide or carbon monoxide And mixing the mixture with an additive such as an excipient into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.
환 형태의 건강기능식품은 본 발명의 유효성분인 일산화탄소 또는 일산화탄소 공급체와 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다.The ring-shaped health functional food can be prepared by molding a mixture of the carbon monoxide or carbon monoxide supply material, the active ingredient of the present invention, excipient, binder, disintegrant and the like by a conventionally known method, It may be applied to the skin, or the surface may be coated with a substance such as starch or talc.
과립 형태의 건강기능식품은 본 발명의 유효성분인 일산화탄소 또는 일산화탄소 공급체와 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.The granular health functional food may be prepared by granulating a carbon monoxide or carbon monoxide feed, which is an effective ingredient of the present invention, with an excipient, a binder, a disintegrant, etc., in a granular form by a conventionally known method, And a mating agent.
본 발명의 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 건강기능식품은 골 손실을 억제하므로 골 손실로 야기되는 골대사 질환 예방 또는 개선에 효과적이다. 본 발명의 골대사 질환은 골다공증, 파제트 골질환, 구루병, 골연화증, 퇴행성 골질환등과 같은 질환일 수 있으나, 가장 바람직하게는 폐경기 후 골 손실에 따른 골다공증일 수 있다.The health functional food containing the carbon monoxide or carbon monoxide supply body of the present invention as an effective ingredient suppresses bone loss and is therefore effective for preventing or improving bone metabolic diseases caused by bone loss. The bone metabolic diseases of the present invention may be diseases such as osteoporosis, Paget's bone disease, rickets, osteomalacia, degenerative bone disease and the like, and most preferably osteoporosis due to bone loss after menopause.
상기 건강기능식품은 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등일 수 있다.The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.
본 발명은 또한 골 손실 억제용 의약 또는 식품의 제조를 위한 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 조성물의 용도를 제공한다. 상기한 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 본 발명의 조성물은 골 손실과 관련된 질환의 예방 또는 치료용 의약 또는 식품의 제조를 위한 용도로 이용될 수 있다.The present invention also provides the use of a composition comprising, as an active ingredient, a carbon monoxide or carbon monoxide supply body for the production of a medicament for inhibiting bone loss or food. The composition of the present invention containing the above-described carbon monoxide or carbon monoxide supply material as an active ingredient can be used for the manufacture of medicines or foods for the prevention or treatment of diseases related to bone loss.
또한 본 발명은 포유동물에게 일산화탄소 또는 일산화탄소 공급체를 투여하는 것을 포함하는 골 손실에 의한 골대사 질환 예방 또는 치료방법을 제공한다.The present invention also provides a method of preventing or treating bone metabolic diseases caused by bone loss, which comprises administering carbon monoxide or carbon monoxide supply to a mammal.
여기에서 사용된 용어 "포유동물"은 치료, 관찰 또는 실험의 대상인 포유동물을 말하며, 바람직하게는 인간을 말한다. The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.
여기에서 사용된 용어 "치료상 유효량"은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다. 본 발명의 유효 성분에 대한 치료상 유효 투여량 및 투여횟수는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로, 투여될 최적의 투여량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효성분 및 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 본 발명의 치료방법에 있어서, 성인의 경우, 본 발명의 일산화탄소 또는 일산화탄소 공급체의 바람직한 전체 용량은 1일당 환자 체중 1 ㎏ 당 약 0.01 ㎍ 내지 1,000 mg, 가장 바람직하게는 0.1 ㎍ 내지 100 mg일 수 있다.The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, the preferred total dose of the carbon monoxide or carbon monoxide supply of the present invention for an adult may be from about 0.01 μg to 1,000 mg, and most preferably from 0.1 μg to 100 mg per kg body weight of the patient per day have.
본 발명의 치료방법에서 본 발명의 일산화탄소 또는 일산화탄소 공급체를 유효성분으로 포함하는 조성물은 경구, 직장, 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 국소, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다.
In the treatment method of the present invention, the composition comprising the carbon monoxide or carbon monoxide supply material of the present invention as an active ingredient may be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, topically, In a conventional manner.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예들은 본 발명을 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.
<< 실시예Example > >
통계처리Statistical processing
실험결과는 평균±표준편차로 표기하였고, Student's t-test로 분석하여 p 값이 0.05 미만일 때 통계적으로 유의하다고 판단하였다.
The test results were expressed as mean ± SD, analyzed by Student's t-test, and statistically significant when the p-value was less than 0.05.
<< 실시예Example 1> 1>
일산화탄소에 의한 파골세포 분화 억제 효과 (Inhibitory Effect of Carbon Monoxide on Osteoclast Differentiation
inin
vitrovitro
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<1-1> 마우스 골수세포의 배양<1-1> Culture of mouse bone marrow cells
4-5주령의 C57BL/6J 마우스를 경추 탈골한 후 70% 에탄올로 소독하였다. 대퇴골 및 경골부분의 피부를 절개하여 부착 근육을 떼어냈다. 경골 원심부를 절단하고 슬개골을 탈골시켜 경골을 적출하였다. 뼈 양끝을 조금 잘라 한 쪽 끝에 21-gauge 주사바늘을 꽃고 α-MEM을 흘려보내 골수세포를 시험관에 모았다. 원심 분리한 후 α-MEM에 현탁하고 2배의 Gey’s solution을 가해 적혈구를 제거했다. 원심 분리한 후 모아진 골수세포를 10% FBS가 함유된 α-MEM으로 재현탁한 후 배양하여 사용하였다.
4-5 week old C57BL / 6J mice were dismissed from the cervical vertebra and disinfected with 70% ethanol. The skin of the femur and tibia was dissected and the attachment muscles were removed. The distal tibia was cut and the patella was dislocated to remove the tibia. The bone marrow cells were collected in vitro by flowing α-MEM through a 21-gauge injection needle at one end of the bone and cutting the bone slightly. After centrifugation, the cells were suspended in? -MEM and 2 times of Gey's solution was added to remove the red blood cells. After centrifugation, the collected bone marrow cells were resuspended in α-MEM containing 10% FBS and cultured.
<1-2> 파골세포의 분화유도 및 분화된 파골세포 판정<1-2> Induction of osteoclast differentiation and differentiation of osteoclast
상기 실시예<1-1>에 따라 초기 배양된 골수세포에 대식세포증식자극인자(macrophage colony stimulating factor : 이하 간략하게 ‘M-CSF’이라 함) 20ng/ml를 첨가하여 16시간 동안 배양하였다. 부유세포들을 수득하여 M-CSF 20ng/ml로 2일 이상 추가 배양하여 골수유래 대식세포(bone marrow-derived macrophage: BMM: 이하 간략하게 ‘BMM’라 함)를 생성시켰고, 생성된 BMM를 회수한 다음 1×105 cells/well에 파골세포분화인자 RANKL(receptor activator of NF-κB ligand) 40ng/ml과 M-CSF 20ng/ml를 포함하는 배지를 첨가한 후 배양하였으며, 3일째 배지를 교체하였다. 배양이 끝난 세포는 10% 포르말린으로 10분간 고정한 후 에탄올-아세톤 (1:1)로 1분간 재고정하여 TRAP (tartrate-resistant acid phosphatase, 이하 간략하게 ‘TRAP’라 함) 염색하였고, TRAP-양성 다핵세포 (MNC)의 세포수를 측정하였다.
20 ng / ml of macrophage colony stimulating factor (hereinafter abbreviated as 'M-CSF') was added to the bone marrow cells cultured according to the above Example <1-1> and cultured for 16 hours. The suspension cells were further cultured for 2 days or more with 20 ng / ml of M-CSF to generate bone marrow-derived macrophages (hereinafter simply referred to as BMMs), and the generated BMMs were recovered Then, the medium containing 40 ng / ml of the osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) and 20 ng / ml of M-CSF was added to 1 × 10 5 cells / well and the medium was changed on the third day . The cultured cells were fixed with 10% formalin for 10 minutes, stained with TRAP (tartrate-resistant acid phosphatase, hereinafter simply referred to as TRAP), and stained with ethanol-acetone (1: Cells (MNC) were counted.
<1-3> 일산화탄소 공급체 처리에 따른 파골세포 분화 억제 효과≪ 1-3 > Inhibitory effect on osteoclast differentiation by carbon monoxide supplier treatment
본 발명의 유효성분인 일산화탄소가 파골세포 분화에 어떠한 영향을 미치는지 알아보기 위하여, 파골세포 분화과정에 일산화탄소를 공급할 수 있는 일산화탄소 공급체를 처리한 후 파골세포 분화 억제 정도를 TRAP+(TRAP 양성 다핵세포) 세포의 개수 측정을 통해 확인하였다. 파골세포의 분화가 있을 경우 다핵세포의 개수가 늘어날 것이므로 TRAP+ 세포의 수가 증가하기 때문이다. In order to investigate the effect of carbon monoxide, which is an effective component of the present invention, on the osteoclast differentiation, a carbon monoxide supplier capable of supplying carbon monoxide to the osteoclast differentiation process was treated with TRAP + (TRAP-positive polynuclear cell) The number of cells was confirmed by counting. When osteoclast differentiation occurs, the number of pluripotent cells increases and the number of TRAP + cells increases.
상기 실시예<1-1>에 따라 초기 배양된 골수세포를 M-CSF 20ng/ml로 16시간 배양한 후 부유세포를 M-CSF 20ng/ml로 3일간 추가 배양하고, 생성된 BMM를 회수한 다음 1×105 cells/well에 RANKL 40ng/ml 과 M-CSF 20ng/ml 존재 하에 일산화탄소 공급체인 CORM2(Tricarbonyldichlororuthenium(II) dimmer, [Ru(CO)3Cl2]2)를 농도별(5, 10, 20, 30μM)로 첨가하여 4일간 배양시킨 후, TRAP+ 다핵세포(multinucleate cell: MNC) 수를 측정하였다. 대조군으로는 일산화탄소를 공급하지 못하는 비활성 일산화탄소 공급체(iCORM2)를 사용하였다.The bone marrow cells that were initially cultured according to the above Example <1-1> were cultured with 20 ng / ml of M-CSF for 16 hours. Then, the floating cells were further cultured for 3 days with 20 ng / ml of M-CSF and recovered BMM Next, the carbon monoxide supply CORM2 (Tricarbonyldichlororuthenium (II) dimmer, [Ru (CO) 3 Cl 2 ] 2 ) was added to 1 × 10 5 cells / well in the presence of 40ng / ml RANKL and 20ng / ml M- 10, 20, and 30 μM) and cultured for 4 days. The number of TRAP + multinucleate cells (MNC) was measured. An inactive carbon monoxide feed (iCORM2), which does not supply carbon monoxide, was used as a control.
그 결과 도 1에서는 본 발명의 일산화탄소 공급체(CORM2)를 투입한 군에서 TRAP+ 세포가 농도 의존적으로 감소되는 것을 확인할 수 있었다. 반면에 일산화탄소를 방출할 수 없는 비활성 일산화탄소 공급체(iCORM)를 투입한 군에서는 TRAP+ 세포수를 거의 감소시키지 못하는 것으로 나타났다.
As a result, in FIG. 1, it was confirmed that TRAP + cells were decreased in a concentration-dependent manner in the group to which the carbon monoxide feeder (CORM2) of the present invention was added. On the other hand, in the group injected with an inactive carbon monoxide feeder (iCORM) which can not release carbon monoxide, it was found that the number of TRAP + cells was not substantially reduced.
<1-4> 일산화탄소 공급체 처리가 파골세포 특이적 ≪ 1-4 > Carbonmonoxide feedstock-specific osteoclast- 마커Marker 유전자의 발현에 미치는 영향 Effect on gene expression
일산화탄소가 파골세포 분화를 억제할 수 있음을 더욱 확실히 증명하기 위하여, 일산화탄소의 처리 후 파골세포 특이적 마커 유전자인 TRAP 및 칼시토닌 수용체의 발현을 quantitative PCR을 이용하여 조사하였다.In order to further demonstrate that carbon monoxide could inhibit osteoclast differentiation, the expression of TRAP and calcitonin receptor, an osteoclast - specific marker gene, after carbon monoxide treatment was investigated using quantitative PCR.
상기 실시예<1-1>에 따라 초기 배양된 골수세포를 M-CSF 20ng/ml로 16시간 배양한 후 부유세포를 M-CSF 20ng/ml로 3일간 추가 배양하였다. 생성된 BMM를 회수한 다음 1×105 cells/well에 RANKL 40ng/ml 과 M-CSF 20ng/ml 존재 하에 일산화탄소 공급체인 CORM2(Tricarbonyldichlororuthenium(II) dimmer, [Ru(CO)3Cl2]2)를 30μM농도로 첨가하여 48시간 배양시킨 후, TRAP 및 칼시토닌 수용체(calcitonin receptor)의 발현량을 quantitative PCR를 이용하여 측정하였다. 이때 상기 PCR을 위해 배양된 세포로부터 총 RNA를 추출하였고(인비트로젠사의 제공방밥으로 수행함), 총 RNA는 제조자의 지시에 따라 올리고-dT 및 Supercript Ι enzyme(Invitrogen, Carlsbad, CA)을 이용하여 역전사하였다. qPCR은 SYBR Green 1 Tag polymerase(Qiagen, Hilden, Germany) 및 DNA Engine Opticon Continuous Fluorescence detection system(MJ Research Inc., Waltham, MA) 상에 적절한 프라이머를 이용하여 이행하였고, 이때 각각의 특정 프라이머 세트는 melting curve 분석 및 아가로스젤 전기영동으로써 확인하여 특이성을 확인하였으며, 사용한 프라이머 서열은 표 1에 기재하였다. 또한 housekeeping 유전자인 GAPDH 유전자를 대조군으로 사용하였다. GAPDH와 비교하여 상대적인 카피 수는 2-△△ Ct를 이용하여 계산되었다(Livak, K.J., and T.D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-Ctmethod.Methods.25:402-408.). 프라이머 서열은 아래의 서열을 이용하였다.
The bone marrow cells that were initially cultured according to the above Example <1-1> were cultured with 20ng / ml of M-CSF for 16 hours, and the floating cells were further cultured with 20ng / ml of M-CSF for 3 days. The resulting BMM was recovered and then treated with CORM2 (Tricarbonyldichlororuthenium (II) dimmer, [Ru (CO) 3 Cl 2 ] 2 ) at 1 × 10 5 cells / well in the presence of 40ng / ml RANKL and 20ng / ml M- Was added at 30 μM concentration for 48 hours. The expression level of TRAP and calcitonin receptor was measured by quantitative PCR. At this time, total RNA was extracted from the cells cultured for the PCR (performed as provided by Invitrogen), and the total RNA was quantified using oligo-dT and SuperScript I enzyme (Invitrogen, Carlsbad, CA) And reversed. qPCR was performed using the appropriate primers on the
(calcitonin receptor)Calcitonin receptor
(calcitonin receptor)
그 결과 도 2에서 나타난 바와 같이, TRAP 및 칼시토닌 수용체의 전사체(transcripts)는, 본 발명의 일산화탄소 공급체인 CORM2를 투입한 군에서 상당히 감소되는 것으로 나타났다. 이를 통해 파골세포 특이적 마커 유전자의 발현은 일산화탄소에 의해 발현이 억제됨을 확인할 수 있었으며, 궁극적으로 파골세포 분화를 감소시킬 수 있음을 의미하는 것이다.
As a result, transcripts of TRAP and calcitonin receptors were shown to be significantly reduced in the group injected with the carbon monoxide supply CORM2 of the present invention, as shown in Fig. This suggests that expression of osteoclast-specific marker genes is inhibited by carbon monoxide, which ultimately reduces osteoclast differentiation.
<1-5> 일산화탄소의 파골세포 형성 활성 분석<1-5> Analysis of osteoclast formation activity of carbon monoxide
본 발명의 유효성분인 일산화탄소가 파골세포의 형성을 억제하는 것을 상기의 실험을 통해 증명하였다. 이러한 일산화탄소의 파골세포의 형성 억제 기작이 파골세포의 형성 초기단계에 일어나는지 아니면 성숙한 파골세포에서도 일어날 수 있는지 규명하기 위해 파골세포 형성 단계별 일산화탄소 공급체(CORM2)를 투입하여 파골세포 억제 정도를 실험하였다.The above experiment proved that carbon monoxide, which is an effective ingredient of the present invention, inhibits osteoclast formation. In order to investigate whether the inhibition mechanism of osteoclast formation of osteoclasts occurs in the early stage of osteoclast formation or in osteoclast, osteoclast suppression level of osteoclast formation step (CORM2) was experimentally examined.
상기 실시예<1-1>에 따라 초기 배양된 골수세포를 M-CSF 20ng/ml로 16시간 배양한 후 부유세포를 M-CSF 20ng/ml로 2일간 추가 배양하였다. 생성된 BMM를 회수한 다음 1×105 cells/well에 RANKL 40ng/ml 과 M-CSF 20ng/ml 존재 하에 일산화탄소 공급체인 CORM2(Tricarbonyldichlororuthenium(II) dimmer, [Ru(CO)3Cl2]2)를 30μM농도로 시간대별(0-4D : 0일 투입후 96 h 지속, 0-2D : 0일 투입후 48 h에 제거, 3-4D: 49h 투입후 96 h 지속)로 투입하였다. 막대그래프위의 수는 각 처리군의 TRAP-positive MNC를 CORM2로 처리하지 않은 세포의 TRAP-positive MNC로 나눈 수치이다(CORM2에 의한 억제에 반비례).The bone marrow cells initially cultured according to the above Example <1-1> were cultured for 16 hours with 20ng / ml of M-CSF, and the floating cells were further cultured with 20ng / ml of M-CSF for 2 days. The resulting BMM was recovered, and then a carbon monoxide supply CORM2 (Tricarbonyldichlororuthenium (II) dimmer, [Ru (CO) 3Cl 2] 2) was added to 1 × 10 5 cells / well in the presence of 40ng / ml of RANKL and 20ng / ml of M- (0-4D: 0 day, 96h continuous, 0-2D: 0 day, 48h removal, 3-4D: 49h addition, 96h continuous). The number on the bar graph is the number of TRAP-positive MNCs in each treatment group divided by TRAP-positive MNCs in cells not treated with CORM2 (inversely proportional to inhibition by CORM2).
그 결과 도 3에서 나타난 바와 같이 CORM2는 파골세포형성 초기단계에서 일산화탄소 형성의 저해가 더 효율적인 것으로 나타났다.As a result, as shown in Fig. 3, CORM2 showed more inhibition of carbon monoxide formation at the early stage of osteoclast formation.
그러므로 CORM2의 파골세포 분화 억제 효과는 분화의 초기단계에서 더 효과적일 수 있음을 알 수 있었다.
Therefore, the inhibitory effect of CORM2 on osteoclast differentiation could be more effective in the early stage of differentiation.
<< 실시예Example 2> 2>
일산화탄소의 파골세포 분화 억제 기전 규명 (Identification of the inhibitory mechanism of osteoclast differentiation by carbon monoxide inin vitrovitro ))
파골세포 분화에 관한 RANKL 자극을 매개하는 다양한 신호전달 경로가 밝혀진바 있다. 특히 RANKL의 자극은 파골전구세포에서 파골세포의 분화를 조절하는 핵심 전사인자로 알려져 있는 NFAT(nuclear factor of activated T cells)의 발현을 유도한다. NFAT는 RANKL 자극에 의한 세포내 칼슘 신호 전달로 초기 유도되며, 그 후 ERK(extracellular signal-regulated kinase), NF-κB, MAPK(p38 MAP kinase), c-Fos 등의 활성화가 NFAT의 자가증폭(autoamplification)을 유발하여 활성이 지속된다. 핵심 전사인자인 NFAT 단백질은 현재까지 5 종류가 확인되었다. 즉, NFAT family는 NFAT1 (NFATp 또는 NFATc2), NFAT2 (NFATc 또는 NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx 또는 NFATc3) 및 NFAT5으로 구성되어 있다.Various signaling pathways mediating RANKL stimulation on osteoclast differentiation have been identified. In particular, RANKL stimulation induces the expression of NFAT (nuclear factor of activated T cells), a key transcription factor that regulates osteoclast differentiation in osteoclast precursor cells. NFAT was initially induced by intracellular calcium signaling by RANKL stimulation and then activation of ERK (extracellular signal-regulated kinase), NF-κB, MAPK (p38 MAP kinase) autoamplification) and the activity continues. NFAT protein, a key transcription factor, has been identified to date. That is, the NFAT family consists of NFAT1 (NFATp or NFATc2), NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3) and NFAT5.
본 발명의 일산화탄소가 RANKL에 의한 파골세포 분화와 이에 따른 골 흡수 억제할 수 있음을 상기 실시예를 통해 밝혔으므로, 본 실험에서는 일산화탄소가 파골세포 전구인자에서 RANKL의 자극에 의해 유도된 신호 경로의 활성에 영향을 주는지를 실험하였다.Since the carbon monoxide of the present invention has been shown to be able to inhibit osteoclast differentiation by osteoclast differentiation and thus to inhibit bone resorption, in this experiment, it was shown that carbon monoxide inhibited the activity of the signal pathway induced by RANKL stimulation in osteoclast precursor factors Were examined.
따라서 작용기전을 규명하기 위하여 첫 번째, 일산화탄소의 처리가 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT2의 발현 및 c-fos의 발현에 미치는 영향을 조사하였다. 또한 두 번째, NF-κB의 활성화에 미치는 영향을 조사하였다. 마지막으로 세 번째, NFAT2 활성을 면역형광 염색(항-NEAF2 항체)법을 수행하여 측정하였다.
Therefore, the effects of carbon monoxide treatment on the expression of NFAT2 and the expression of c-fos, which are key regulators of osteoclast differentiation, were investigated. Secondly, the effect on NF-κB activation was investigated. Finally, third, NFAT2 activity was measured by immunofluorescence staining (anti-NEAF2 antibody) method.
<2-1> 일산화탄소 공급체 처리에 따른 ≪ 2-1 > NFAT2NFAT2 및 c- And c- fosfos 의 발현 측정Of expression
본 발명의 일산화탄소의 처리가 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT2의 발현 및 c-fos의 발현에 미치는 영향을 조사하였다.The effects of the carbon monoxide treatment of the present invention on the expression of NFAT2 and c-fos expression, which are key transcription factors controlling osteoclast differentiation, were investigated.
BMM 세포를 RANKL 40ng/ml 과 M-CSF 20ng/ml 존재 하에 일산화탄소 공급체인 CORM2(Tricarbonyldichlororuthenium(II) dimmer, [Ru(CO)3Cl2]2)를 30μM농도로 첨가하여 48시간 배양시킨 후, NFAT2 및 c-fos의 발현량을 quantitative PCR를 이용하여 측정하였다. qPCR은 하기의 프라이머 서열을 사용하여 실시하였으며, 상기 실시예<1-4>와 동일한 방법으로 수행되었다.
BMM cells RANKL 40ng / ml, and M-CSF 20ng / ml under a carbon monoxide feed present chain CORM2 (Tricarbonyldichlororuthenium (II) dimmer, [Ru (CO) 3 Cl 2] 2) After 48 hours of incubation by adding a 30μM concentration, The expression levels of NFAT2 and c-fos were measured by quantitative PCR. The qPCR was performed using the following primer sequences and performed in the same manner as in Example <1-4>.
그 결과 도 4에서 나타난 바와 같이, c-Fos 및 NFAT2 전사체(transcripts)는, 본 발명의 일산화탄소 공급체인 CORM2를 투입한 군에서 상당히 감소되는 것으로 나타났다. 이상의 결과를 통해 일산화탄소는 NFAT2 및 c-Fos의 발현 감소를 통하여 파골세포 분화를 억제한다는 사실을 알 수 있었다.
As a result, as shown in Fig. 4, the c-Fos and NFAT2 transcripts were significantly reduced in the group to which the carbon monoxide supply CORM2 of the present invention was injected. These results suggest that carbon monoxide inhibits osteoclast differentiation through decreased expression of NFAT2 and c-Fos.
<2-2> 일산화탄소 공급체 처리에 따른 ≪ 2-2 > NFNF -κB의 활성화 측정-K activation assay
본 발명의 일산화탄소 처리가 파골세포의 분화를 조절하는 핵심 전사인자인 NF-κB의 활성화에도 영향을 미치는지 조사하였다. NF-κB는 RANKL 자극에 의해 활성화된 후 핵 내로 들어가 필요한 유전자의 프로모터에 결합하여 해당 유전자 발현을 유도한다. 이때 생성된 각종 단백질은 외부자극에 대한 세포 반응 유발에 관여하게 된다. 따라서 본 발명자들은 일산화탄소에 의한 NF-κB의 활성도를 측정하기 위해 electrophoretic mobility shift assay (EMSA) 방법을 수행하였다.The present inventors also investigated whether the carbon monoxide treatment of the present invention affects the activation of NF-κB, a key transcription factor that regulates osteoclast differentiation. NF-κB is activated by RANKL stimulation, enters the nucleus, binds to the promoter of the required gene, and induces the gene expression. At this time, the generated various proteins are involved in the induction of cell responses to external stimuli. Therefore, the present inventors performed electrophoretic mobility shift assay (EMSA) to measure the activity of NF-κB by carbon monoxide.
BMM 세포를 RANKL 40ng/ml, M-CSF 20ng/ml 존재 하에서 일산화탄소 공급체인 CORM2(Tricarbonyldichlororuthenium(II) dimmer, [Ru(CO)3Cl2]2)를 농도별(20μM, 50μM)로 첨가하여 1시간 동안 배양하였으며, 이렇게 배양된 세포로부터 핵 추출물을 수득하였다. 또한 NF-κB 결합 실험은 NF-κB 일치 결합부위를 포함하는 하기 표 3의 서열번호 11의 이중가닥 올리고뉴클레오타이드(Santa Cruz Biotechnology, San Diego, CA)를 이용하여 수행하였다. 상기 서열번호 11의 NF-κB의 결합 특이성은 하기 표 3의 서열번호 12의 변이체 서열을 사용하여 경쟁 에세이를 통하여 확인할 수 있었다. 서열번호 11의 올리고뉴클레오타이드 또는 서열번호 12의 변이체는은 T4 폴리뉴클레오타이드 키나아제(Promega, Madison, WI)를 이용하여 [γ-32P]로 말단을 표지화시켰다. 각각의 핵 추출물 5μg은 1μg poly(dIdC), 15mM HEPES, pH7.6, 80mM NaCl, 1mM EGTA, 1mM DTT(dithio threitol) 및 10% 글리세롤을 함유하는 결합 버퍼 10μl에 32P-표지화된 프로브 1ng과 함께 20분 동안 30℃에서 반응시켰다. DNA-단백질 결합체들은 native 5% 폴리아크릴아마이드 젤 상에서 전기영동한 후, 진공-건조시키고 80℃에서 identifying screen을 이용하여 자기방사법으로 가시화하였다.BMM cells were treated with CORM2 (Tricarbonyldichlororuthenium (II) dimmer, [Ru (CO) 3 Cl 2 ] 2 ) at concentrations of 20 μM and 50 μM in the presence of 40 ng / ml RANKL and 20 ng / ml M- Hr, and nuclear extracts were obtained from the thus cultured cells. NF-κB binding experiments were also performed using the double-stranded oligonucleotide of SEQ ID NO: 11 (Santa Cruz Biotechnology, San Diego, Calif.), Which contains the NF-κB matching binding site. The binding specificity of NF-κB of SEQ ID NO: 11 was confirmed by competitive assay using the mutant sequence of SEQ ID NO: 12 in Table 3 below. The oligonucleotide of SEQ ID NO: 11 or the variant of SEQ ID NO: 12 was terminally labeled with [? - 32 P] using silver T4 polynucleotide kinase (Promega, Madison, Wis.). 5 μg of each nuclear extract was incubated with 1 ng of 32 P-labeled probe in 10 μl of binding buffer containing 1 μg poly (dIdC), 15 mM HEPES, pH 7.6, 80 mM NaCl, 1 mM EGTA, 1 mM DTT (dithio threitol) and 10% Lt; RTI ID = 0.0 > 30 C < / RTI > DNA-protein conjugates were electrophoresed on native 5% polyacrylamide gels, then vacuum-dried and visualized by self-spinning using a qualifying screen at 80 ° C.
그 결과 도 5에서 나타난 바와 같이, 본 발명의 CORM2를 처리한 군에서는 NF-κB와 DNA의 결합체가 감소된 것을 확인할 수 있었고, 이때 처리한 CORM2의 농도에 비례하여 감소하는 것으로 나타났다(라인 3, 4). 이를 통해 본 발명자들은 CORM2의 처리에 의해 발생되는 일산환탄소는 NF-κB의 활성을 감소시켜 핵 내로의 NF-κB의 이동을 억제하며, 이로 인해 종국에는 NF-κB에 의한 세포신호 전달을 억제하여 파골세포 분화를 억제한다는 것을 알 수 있었다.
As a result, as shown in FIG. 5, it was confirmed that the conjugate of NF-κB and DNA was reduced in the group treated with CORM2 of the present invention, and decreased in proportion to the concentration of treated CORM2 (
올리고뉴클레오타이드NF-B matching site
Oligonucleotide
<2-3> 일산화탄소 공급체 처리에 따른 <2-3> According to carbon monoxide supplier treatment NEAF2NEAF2 활성 실험 Active experiment
또한 본 발명의 일산화탄소 처리에 따른 RANKL에 의해 유도된 파골세포 분화 과정에서 NFAT2 활성을 가시적으로 확인하기 위하여 면역형광 염색(항-NFAT2 항체)에 의하여 실험하였다.In order to confirm the NFAT2 activity in RANKL-induced osteoclast differentiation according to the carbon monoxide treatment of the present invention, immunofluorescence staining (anti-NFAT2 antibody) was performed.
BMM 세포를 RANKL 40ng/ml, M-CSF 20ng/ml 존재 하에서 일산화탄소 공급체인 CORM2(Tricarbonyldichlororuthenium(II) dimmer, [Ru(CO)3Cl2]2)를 30μM농도로 첨가하여 3일 동안 배양하였다. 이 세포들을 수득하여 10%의 중성 버퍼 포르말린으로 고정하였으며, 0.1% 트리톤 X-100을 이용하여 침투시켰다. 면역형광 염색은 FITC-컨쥬게이티드 항-마우스 IgG(eBioscience)를 따라서 마우스 항-NFAT2 항체 (Santa Cruz Biotechnology)를 이용하여 수행되었다. FITC-라벨링된 NFAT2의 subcellular localization은 공초점 레이저 현미경(Olympus FV500)을 이용하여 확인되었다.And the carbon monoxide feed chain CORM2 (Tricarbonyldichlororuthenium (II) dimmer, [Ru (CO) 3 Cl 2] 2) was added to a 30μM concentration of the BMM cells under RANKL 40ng / ml, M-CSF 20ng / ml there were cultured for 3 days. The cells were harvested and fixed with 10% neutral buffered formalin and infiltrated with 0.1% Triton X-100. Immunofluorescent staining was performed using mouse anti-NFAT2 antibody (Santa Cruz Biotechnology) following FITC-conjugated anti-mouse IgG (eBioscience). Subcellular localization of FITC-labeled NFAT2 was confirmed using a confocal laser microscope (Olympus FV500).
그 결과 도 6에서 나타난 바와 같이, RANKL 무처리 군에서는 NFAT2 염색은 거의 없는 것을 발견할 수 있었으며(왼쪽), 반면에 RANKL만을 처리한 군에서는, NFAT2는 다핵 성숙 파골세포로의 핵 이동이 관찰되었다(오른쪽). 또한 본 발명의 일산화탄소 공급체인 CORM2를 함께 처리한 파골세포에서 NFAT2 염색은, RANKL만을 처리한 군(CORM2 무처리)과 비교하여 낮은 수준을 나타내는 것을 확인할 수 있었다(중간).
As a result, as shown in Fig. 6, it was found that RANKL-untreated group showed almost no NFAT2 staining (left), whereas in the RANKL treated group, NFAT2 showed nuclear migration to polynuclear oocyte osteoclast (Right side). In addition, it was confirmed that NFAT2 staining in the osteoclasts treated with the carbon monoxide supply chain CORM2 of the present invention was lower than that of the group treated with only RANKL (CORM2 untreated) (middle).
상기의 실험 결과를 통해 BMM(골수유래 대식세포)에 본 발명의 일산화탄소 공급체인 CORM2를 처리하였을 때 감소된 파골세포형성은 RANKL의 자극에 의해 유도된 신호 경로에서 NFAT2와 c-fos의 발현 억제 및 NF-κB의 감소된 활성과 연계된 것으로 판단하였다. 이는 일산화탄소의 골 대사에서의 특별한 역할을 규명한 것이다.
As a result of the above-mentioned experimental results, when BMM (bone marrow-derived macrophage) was treated with the carbon monoxide supply CORM2 of the present invention, reduced osteoclast formation inhibited the expression of NFAT2 and c-fos in the signal pathway induced by RANKL stimulation NF-κB, respectively. This is the specific role of carbon monoxide in bone metabolism.
<< 실시예Example 3> 3>
일산화탄소의 파골세포 분화에 대한 효능 검증 (Efficacy of Carbon Monoxide on Osteoclast Differentiation inin vivovivo ))
일산화탄소가 in vitro에서 파골세포 분화 억제 효과를 보였으므로 난소절제술을 시행한 마우스 모델을 이용해 일산화탄소의 in vivo 효능을 검사하였다. Carbon monoxide was shown to inhibit osteoclast differentiation in vitro, so the in vivo efficacy of carbon monoxide was examined using an ovariectomized mouse model.
본 발명자들은 일산화탄소가 폐경기 이후에 발생하는 골 손실을 개선시킬 수 있는지 확인하기 위해, 폐경기의 증상이 나타나는 마우스 모델을 제조하기 위해 난소가 적출된 마우스를 제조하였다. 이러한 마우스 모델을 위해 6주령의 C57BL/6J(B/6) 마우스를 대상으로 수술(난소절제술)후 2일째에 일산화탄소 공급체CORM2(5mg/kg), 비활성 일산화탄소 공급체 iCORM2(5mg/kg), 및 베히클(대조군) 가각을 복강에 주입시키고 8주 동안 사육하였다. 모든 마우스들을 대상으로 하는 실험들은 동물보호협회, 울산대학교 협회 및 면역조절 리서치 센타에서 승인된 방법에 따라 수행하였다. 상기의 방법으로 준비된 마우스들은 분석 전에 희생되었으며, 마우스들의 대퇴골은 micro CT로 단층 촬영하여 골 손실의 정도를 확인하였다. 실험동물의 대퇴골은 GE eXplore Locus SP system에 의해 수행되어지는 Micro CT scanning에 의하여 분석되었다. 3차원 histomorphometry 및 긴 뼈 구조의 가시화를 위하여 마우스들의 대퇴골은 high resolution micro-CT 이미지 시스템(0.008-mm 효율적인 탐지기 픽셀 사이즈가 탑재된)에서 스캔되었는데, 3D 마이크로구조 분석의 소프트프로그램은 explore MicroView 2.2.로부터 제공되었다. To confirm whether carbon monoxide could improve bone loss occurring after menopause, the present inventors prepared ovariectomized mice to prepare a mouse model in which symptoms of menopausal symptoms appeared. For this mouse model, C57BL / 6J (B / 6) mice at 6 weeks of age were treated with carbon monoxide supplier CORM2 (5mg / kg), inactive carbon monoxide supplier iCORM2 (5mg / kg) And Beckik (control) were injected into the abdominal cavity and fed for 8 weeks. Experiments involving all mice were performed according to methods approved by the Animal Protection Association, the University of Ulsan, and the Immunomodulatory Research Center. Mice prepared by the above method were sacrificed before analysis, and the femur of mice was examined by micro CT to confirm the degree of bone loss. The femur of the experimental animals was analyzed by Micro CT scanning performed by GE eXplore Locus SP system. For 3D histomorphometry and visualization of long bone structures, the femurs of the mice were scanned in a high resolution micro-CT imaging system (with an effective detector pixel size of 0.008-mm). Lt; / RTI >
그 결과는 하기 도 7 내지 도 10에서 나타내었다.The results are shown in Figs. 7 to 10 below.
도 7은 실험대상 마우스들의 대퇴골을 micro CT를 이용하여 단층 촬영한 사진으로, 이를 자세히 살펴보면 상부와 하부는 각각 마우스 대퇴골의 성장판으로부터 0.7mm 및 2.3mm 부분과 일치하는 부분으로, 사진에서 보이는 바와 같이 베히클을 투입한 군과 비교하여 본 발명의 CORM2를 투입한 군에서 높은 골량을 나타내는 것을 육안으로 확인할 수 있었다.FIG. 7 is a photograph of the femur of the subject's mouse using micro CT. As shown in FIG. 7, the upper and lower portions correspond to the 0.7 mm and 2.3 mm portions from the growth plate of the mouse femur, respectively. Compared with the group to which Beckl were introduced, it was visually confirmed that the group having CORM2 of the present invention showed a high bone mass.
또한 도 8은 실험대상 마우스들의 대퇴부의 해면골의 골량 증가를 수치로 나타낸 그래프로, 이 때 골량 증가는 대퇴골 원위(distal femur)의 secondary spongia의 수치로 평가되었다. 도 8에서 나타는 바와 같이 베히클을 투입한 군과 비교하여 본 발명의 CORM2를 투입한 군에서 골밀도(BMD:Bone Mineral Density)가 상당히 높은 것을 확인할 수 있었다.FIG. 8 is a graph showing numerical increase in bone mass of the spongy bone of the femur of the subject mice, wherein the increase in bone mass was evaluated as the secondary spongia of the distal femur. As shown in FIG. 8, it was confirmed that BMD (bone mineral density) was significantly higher in the group to which CORM2 of the present invention was added compared with the group to which Beckle was added.
또한 도 9는 뼈의 무기질함량(BMC: bone mineral content)을, 도 10은 전체 조직 볼륨의 퍼센트로써 표현된 해면골 부피(BV/TV %)를 나타낸 것으로, 상기 도 8과 유사한 패턴으로 나타났다. 즉 여기에서도 베히클을 투입한 군과 비교하여 본 발명의 CORM2를 투입한 군에서 뼈의 무기질함량 및 해면골 부피가 상당히 높은 것을 확인할 수 있었다.
Fig. 9 shows the bone mineral content (BMC) and Fig. 10 shows the cancellous bone volume (BV / TV%) expressed as a percentage of the total tissue volume. In other words, it was confirmed that the mineral content of the bone and the cancellous bone volume were significantly higher in the group to which the CORM2 of the present invention was added, as compared with the group to which Beckl were added.
상기의 실시예 1 내지 3을 종합해 보면, 파골세포로 분화 과정 중인 세포에 본 발명의 일산환탄소를 처리한 경우 파골세포로의 형성이 억제되는 것이 증명되었으며; 이러한 파골세포의 형성을 억제하는 기작은 RANKL의 자극에 의해 유도된 분자적 신호경로(signal pathway)에서 파골세포 분화를 조절할 수 있는 핵심 전사인자인 NFAT2와 c-fos의 발현 억제 및 NF-κB의 감소된 활성과 연계되어 있으며; 마지막으로 난소절제술을 시행한 마우스 모델에 일산화탄소 공급체(CORM2)를 투입한 경우 골밀도, 뼈의 무기질함량 등의 증가를 확인함으로써 상기 in vitro 실험과 동일한 결과를 도출할 수 있었다. 따라서 상기와 같은 특징을 가진 일산화탄소를 이용하는 경우 폐경기 이후에 발생하는 골 손실을 개선에 효과적일 것으로 판단되었다.
In the above Examples 1 to 3, when osteoclast carbon of the present invention was treated with osteoclast-differentiating cells, formation of osteoclasts was inhibited; The mechanism of this inhibition of osteoclast formation is to inhibit the expression of NFAT2 and c-fos, the key transcription factors that regulate osteoclast differentiation in the molecular signal pathway induced by RANKL stimulation, and to inhibit NF-κB Associated with reduced activity; Lastly, when the carbon monoxide feeder (CORM2) was injected into the ovariectomized mouse model, the increase in the bone mineral density and the bone mineral content was confirmed, and the same results as those of the in vitro experiment could be obtained. Therefore, when using the carbon monoxide having the above characteristics, it would be effective to improve bone loss occurring after menopause.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
<110> University of Ulsan Foundation For Industry Cooperation <120> Composition Comprising Carbon Monoxide or Carbon Monoxide Donor for Inhibition of Bone Loss <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> calcitonin receptor forward primer <400> 1 ctgctcctag tgagcccaac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> calcitonin receptor reverse primer <400> 2 cagcaatcga caaggagtga 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TRAP forward primer <400> 3 gaccaccttg gcaatgtctc tg 22 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TRAP reverse primer <400> 4 tggctgagga agtcatctga gttg 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 5 acccagaaga ctgtggatgg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 6 cacattgggg gtaggaacac 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> c-fos forward primer <400> 7 agcctttcct actaccattc cc 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-fos reverse primer <400> 8 tggcactaga gacggacaga 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NFAT2 forward primer <400> 9 aataacatgc gagccatcat c 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NFAT2 reverse primer <400> 10 tcaccctggt gttcttcctc 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NF-kB specific binding oligonucleotide <400> 11 agttgagggg actttcccag gc 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mutant of NF-kB specific binding oligonucleotide <400> 12 agttgaggcg actttcccag gc 22 <110> University of Ulsan Foundation for Industry Cooperation <120> Composition Comprising Carbon Monoxide or Carbon Monoxide Donor for Inhibition of Bone Loss <160> 12 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> calcitonin receptor forward primer <400> 1 ctgctcctag tgagcccaac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> calcitonin receptor reverse primer <400> 2 cagcaatcga caaggagtga 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TRAP forward primer <400> 3 gaccaccttg gcaatgtctc tg 22 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TRAP reverse primer <400> 4 tggctgagga agtcatctga gttg 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 5 acccagaaga ctgtggatgg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 6 cacattgggg gtaggaacac 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> c-fos forward primer <400> 7 agcctttcct actaccattc cc 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-fos reverse primer <400> 8 tggcactaga gacggacaga 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NFAT2 forward primer <400> 9 aataacatgc gagccatcat c 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NFAT2 reverse primer <400> 10 tcaccctggt gttcttcctc 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NF-kB specific binding oligonucleotide <400> 11 agttgagggg actttcccag gc 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mutant of NF-kB specific binding oligonucleotide <400> 12 agttgaggcg actttcccag gc 22
Claims (7)
상기 일산화탄소 또는 일산화탄소 공급체는 폐경기로 인해 유발되는 골 손실을 감소시키는 것을 특징으로 하는 골 손실 억제용 약학 조성물.The method according to claim 1,
Wherein the carbon monoxide or carbon monoxide supply body reduces bone loss caused by menopause.
상기 일산화탄소 또는 일산화탄소 공급체는 파골세포의 분화를 조절하는 핵심 전사인자인 NFAT2와 c-fos의 발현 억제 및 NF-κB의 활성화를 억제하는 기전을 통해 골 손실을 감소시키는 활성을 갖는 것을 특징으로 골 손실 억제용 약학 조성물.The method according to claim 1,
The carbon monoxide or carbon monoxide supply agent has an activity of reducing bone loss through a mechanism of inhibiting the expression of NFAT2 and c-fos, which are key transcription factors controlling osteoclast differentiation, and the activation of NF-κB, A pharmaceutical composition for inhibiting loss.
상기 일산화탄소 또는 일산화탄소 공급체는 폐경기로 인해 유발되는 골 손실을 감소시키는 것을 특징으로 하는 골 손실 억제를 위한 건강기능식품.6. The method of claim 5,
Wherein said carbon monoxide or carbon monoxide supply body reduces bone loss caused by menopause.
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