KR101909954B1 - Composition for prevention or treatment of sepsis or septic shock comprising Zingerone - Google Patents
Composition for prevention or treatment of sepsis or septic shock comprising Zingerone Download PDFInfo
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- KR101909954B1 KR101909954B1 KR1020170048759A KR20170048759A KR101909954B1 KR 101909954 B1 KR101909954 B1 KR 101909954B1 KR 1020170048759 A KR1020170048759 A KR 1020170048759A KR 20170048759 A KR20170048759 A KR 20170048759A KR 101909954 B1 KR101909954 B1 KR 101909954B1
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Abstract
Description
본 발명은 진저론을 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 예방 또는 치료용 조성물에 관한 것으로, 보다 상세하게는 진저론을 유효성분으로 포함하는 HMGB1에 의해 매개되는 패혈증 또는 패혈성 쇼크의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating septicemia or septic shock comprising gingerol as an active ingredient, and more particularly to a composition for preventing or treating septicemia or septic shock mediated by HMGB1 comprising gingerol as an active ingredient , ≪ / RTI >
HMGB1(High-mobility group box 1 protein)은 뉴클레오솜(nucleosome)의 구조 및 안정성 유지와 전사 인자들이 그들의 동족 DNA 서열에 결합하는 것을 가능하게 하는 것에 의하여 유전자 발현의 조절에 기여하는 핵 염색체 단백질로서 처음 규명되었다. 최근, 실험 및 임상 연구로부터의 많은 데이터들이, 패혈증과 같은 여러 염증성 질환의 발병에 대한 세포외 HMGB1(extracellular HMGB1)의 기여를 강조하였다(Andersson and Tracey, 2011; Diener et al., 2013). 패혈증 동안, HMGB1은 감염에 기인하는 세포죽음에 따라 급속히 수동적으로 분비될 수 있고, 전-염증성 사이토카인들 및 PAMP(pathogen-associated molecular patterns)들에 대한 반응으로 면역 세포에서 능동적으로 분비될 수 있다. PAMP 및 내생적으로 유래된 염증 매개자(예를들어 TNF-α, IL-1b, NF-κB 및 ERK1/2)에 노출되었을 때, 세포외 산물과 원형질막 수용체의 상호작용에 의하여 촉발되는 세포적 신호 변환(cellular signaling transduction)을 통하여 내피세포는 HMGB1을 능동적으로 분비한다. 세포외 환경(extracellular milieu)으로 분비된 HMGB1은 전-염증성 사이토카인의 생성을 위하여 선천 면역 세포들을 자극하는 케모카인 또는 사이토카인으로서 역할을 한다. HMGB1 상호작용에 참여하는 세포 표면 수용체들은 RAGE(receptor for advanced glycation end products), TLR(toll like receptor)-2, 및 TLR-4를 포함한다. HMGB1이 세포외 공간으로 분비되는 방식은 두가지가 있는데, 하나는 대식세포 및 호중구와 같은 면역세포들이 관련되어 염증을 촉발하는 능동적 프로세스이고, 다른 하나는 괴사성 세포 및 선천 면역 시스템에 의해 채택된 매커니즘에 의하여 촉발되며 손상되거나 괴사된 세포를 인식하는 수동적 프로세스이다(Ulloa and Tracey, 2005). HMGB1으로 내피를 자극하는 것은 ICAM( intercellular adhesion molecule), VCAM(vascular cell-adhesion molecule), 및 E-Selectin과 같은 CMA(cell adhesion molecule)들의 발현을 증가시키며, 이는 백혈구의 소집(recruitment)을 통하여 염증을 촉진한다(Bae and Rezaie, 2011). 패혈증 또는 패혈성 쇼크 진단 후 1주일까지 대부분의 환자에서 측정가능하며, 상기 HMGB1의 수준은 장기 기능이상(organ dysfunction) 정도와 연관되어있다(Gibot et al., 2007; Sunden-Cullberg et al., 2005).HMGB1 (High-
한편, 패혈증(sepsis)은 감염된 미생물에 대항하는 신체의 비정상적인 방어작용에 의해 발생한다. 대식세포의 활성화와 이에 따른 염증관련 인자들의 과도한 생성이 연관되어 있는데, 이로 인해 전신에 심각한 염증 반응이 나타난다. 체온이 38℃ 이상으로 올라가는 발열 증상, 36℃ 이하로 내려가는 저체온증, 분당 24회 이상의 호흡수(빈호흡), 분당 90회 이상의 심박수(빈맥), 혈액 검사상 백혈구 수의 증가 또는 현저한 감소 중 두 가지 이상의 증상을 보이는 경우, 이를 전신성염증반응증후군(systemic inflammatory response syndrome; SIRS)이라하고, 이러한 전신성염증반응증후군이 미생물의 감염에 의한 것일 때 패혈증이라고 한다. 패혈증은 잠재적으로 패혈증성 쇼크(septic shock)를 유발할 수 있다. 패혈증이 심해지면 신체의 여러 기관(심장, 신장, 간, 뇌, 폐 등)의 기능이 나빠지고 더욱 심해지면 쇼크 상태가 되는 것이다. 다양한 종류의 병원체로 인해 패혈증이 발병할 수 있는데, 가장 발생률이 높은 것은 박테리아에 의한 것이지만 그 외에도 바이러스나 곰팡이에의해서도 일어날 수 있다. 폐에 감염을 일으키는 폐렴, 방광과 신장에 감염을 일으키는 요도감염, 피부에 일어나는 봉소염, 복부에 일어나는 충수염 또는 뇌에 일어나는 뇌막염 등이 있으며, 예를 들면 폐렴을 앓고 있는 환자가 패혈증에 걸리게 되면 뇌, 심장, 간, 폐 또는 신장에 손상이 일어나며 중증으로 진전되는 경우 환자의 약 20 ~ 50%는 패혈증성 쇼크에 의해 사망한다. 또한, 수술 후 감염에 의해 패혈증이 발생하기도 한다. 감염 또는 수술 후 감염에 의한 초급성 염증반응으로서 패혈증에 걸리게 되면 40 ~ 90%가 사망에 이르게 된다.Sepsis, on the other hand, is caused by an abnormal defense of the body against the infected microorganism. The activation of macrophages and thus the excessive production of inflammatory factors are associated with a severe inflammatory response in the whole body. Two or more of the following: fever with a temperature above 38 ° C, hypothermia below 36 ° C, at least 24 breaths per minute (ventilation), at least 90 heartbeats per minute (tachycardia) When symptoms are seen, it is called systemic inflammatory response syndrome (SIRS), and it is called sepsis when the systemic inflammatory response syndrome is caused by microbial infection. Septicemia can potentially lead to septic shock. When septicemia gets worse, the function of various parts of the body (heart, kidney, liver, brain, lungs, etc.) deteriorates and becomes more shocked. A variety of pathogens can cause sepsis, the most common of which is caused by bacteria, but can also be caused by viruses or fungi. Pneumonia causing infection of the lungs, urethral infections causing infection of the bladder and kidneys, skin infections, appendicitis in the abdomen, or meningitis in the brain. For example, if a patient with pneumonia is suffering from sepsis, Severe progression of damage to the heart, liver, lungs, or kidneys occurs. Approximately 20-50% of patients die from septic shock. In addition, post-operative infection may cause sepsis. As a hyperinflammatory response due to infection or postoperative infection, 40 to 90% of deaths are caused by sepsis.
상기 패혈증은 감염 원인균과 숙주의 면역, 염증 그리고 응고계통 사이의 복잡한 상호작용의 결과로 발생하는 것으로 이해되고 있다. 숙주의 반응 정도와 감염 원인균의 특성 모두 패혈증의 예후에 중대한 영향을 미친다. 패혈증에서 관찰되는 장기부전은 숙주의 감염 원인균에 대한 반응이 부적절한 경우에 발생하며, 만일 숙주의 감염 원인균에 대한 반응이 지나치게 증폭된다면 숙주 자체의 장기손상을 유발할 수 있다. 이러한 개념을 바탕으로 숙주의 염증 반응에 주도적인 역할을 수행하는 전염증사이토카인(proinflammatory cytokines)인 TNF-α, IL-1β, IL-6등에 대한 길항 물질이 패혈증의 치료제로 시도되었으나 대부분 실패하였으며, 기계환기치료, 활성 단백질 C(activated protein C) 투여, 글루코코르티코이드 치료 등이 현재 시도되고 있으나 여러 가지 한계점이 지적되고 있다. 따라서, 높은 사망률을 보임에도 아직까지 뚜렷한 치료제가 개발되지 않은 패혈증 및 패혈증 쇼크를 예방 또는 치료하기 위한 새로운 치료제에 대한 필요성이 요구되고 있다.It is understood that the sepsis occurs as a result of complex interactions between infectious agents and host immune, inflammation and coagulation systems. Both the response of the host and the characteristics of the causative organism have a significant impact on the prognosis of sepsis. Organ failure observed in sepsis occurs when the host is inadequately reacted to an infectious agent, and if the response to the host's infection causes excessive amplification, it can lead to organ damage in the host itself. Based on this concept, antagonists against proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, which play a leading role in the inflammatory response of the host, , Mechanical ventilation therapy, activated protein C (C) administration, glucocorticoid therapy and the like have been tried, but various limitations have been pointed out. Therefore, there is a need for a new therapeutic agent for preventing or treating septicemia and septic shock, which has not yet developed a clear therapeutic agent even though it shows a high mortality rate.
이에 본 발명자들은 천연물로부터 유래된 항-패혈증 효능 물질을 연구하던 중, 본 명세서에서 진저론(Zingerone)이 패혈증 주요 인자인 HMGB1의 분비를 억제하고 이와 관련된 세포 시그날링의 분자적 매커니즘을 효과적으로 조절하여 HMGB1에 의한 혈관염증성 반응을 억제할 뿐만아니라, 실제로 in vivo 패혈증 동물모델에 대하여 현저한 치료효과를 나타내는 것을 확인하고 본 발명을 완성하였다. Therefore, the inventors of the present invention found that Zingerone inhibits the secretion of HMGB1, which is a major factor of septicemia, and effectively regulates the molecular mechanism of cell signaling associated therewith HMGB1-induced vascular inflammatory response, as well as showing a remarkable therapeutic effect on an animal model of sepsis in vivo, and completed the present invention.
따라서 본 발명의 목적은 하기 화학식 1의 진저론 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 예방 또는 치료용 약학적 조성물을 제공하는 것이다;Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating septicemia or septic shock comprising, as an active ingredient, gingerrone of the following formula (1) or a pharmaceutically acceptable salt thereof:
<화학식 1>≪ Formula 1 >
본 발명의 다른 목적은 진저론 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or ameliorating septicemia or septic shock comprising gingerol or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 본 발명의 목적을 달성하기 위하여, 본 발명은 하기 화학식 1의 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 예방 또는 치료용 약학적 조성물을 제공한다;In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating septicemia or septic shock comprising the compound of the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient;
<화학식 1>≪ Formula 1 >
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 진저론 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 예방 또는 개선용 식품 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a food composition for preventing or ameliorating septicemia or septic shock comprising gingerol or a pharmaceutically acceptable salt thereof as an active ingredient.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 진저론(Zingerone, ZGR) 또는 이의 약학적으로 허용 가능한 염의 신규한 용도를 제공하는 것으로서, 진저론 또는 이의 약학적으로 허용 가능한 염의 항-패혈증 용도를 제공한다;The present invention provides a novel use of Zingerone (ZGR) or a pharmaceutically acceptable salt thereof, which provides anti-septic use of gingerol or a pharmaceutically acceptable salt thereof;
진저론의 항-패혈증 효과는 본 발명자에 의하여 처음으로 규명된 것으로서, 이는 본 발명의 명세서 실시예에 잘 나타나 있다.The anti-septic effect of gingerol was first identified by the present inventor and is well illustrated in the specification examples of the present invention.
본 발명의 실시예에서는, 진저론이 패혈증의 주요 매개자인 HMGB1을 억제함을 확인하였다. In the examples of the present invention, it was confirmed that gingerolone inhibited HMGB1, which is a major mediator of sepsis.
본 발명의 실시예에서는, 진저론이 SIRT1 단백질 발현을 유의하게 증가시켜 HMGB1의 분비를 억제함을 확인하였다.In the examples of the present invention, it was confirmed that gingerrone significantly increased SIRT1 protein expression and suppressed the secretion of HMGB1.
본 발명의 실시예에서는, 패혈증의 주요 매개자인 HMGB1 및 이와 관련된 전염증성 시그날링 인자들에 대하여 진저론의 효과를 평가하였다. In an embodiment of the present invention, the effect of gingerolone was evaluated on HMGB1, a key mediator of sepsis, and proinflammatory signaling factors associated therewith.
진저론은 LPS- 또는 CLP(cecal ligation and puncture)-에 의해 매개된 HMGB1의 분비, HMGB1 수용체(TLR-2, TLR-4 및 RAGE)의 발현 및 HMGB1에 의해 매개된 장벽 붕괴를 억제하였다. 또한 진저론은 CAM들의 발현을 저해하며, HMGB1에 의해 매개된 인간 호중구의 부착 및 이동(이주, migration)을 억제하였다. 게다가 HMGB1에 의해 유도되는 nuclear factor-κB (NF-κB) 및 extracellular regulated kinases 1/2(ERK1/2)의 활성화와 IL-6, tumor necrosis factor-α(TNF-α)의 생산을 억제하였으며, HMGB1으로 자극시, NF-kB p65가 세포질에서 핵으로 전이되는 것을 억제하였다. 또한 상기 화합물들은 실제로 CLP-패혈증 동물모델에서 혈관 장벽의 보호효과를 나타내었으며, 사망률 감소면에서 현저한 효과를 나타내었으며, CLP 수술로 인한 간질 부종, 폐 손상, 조직손상, 간 및 신장에서의 다발성 장기부전, 패혈증 중증 환자에게서 나타나는 내분비계 장애에도 현저한 치료효과가 있다.Ginger Ron inhibited the secretion of HMGB1 mediated by LPS- or CLP (cecal ligation and puncture), the expression of HMGB1 receptors (TLR-2, TLR-4 and RAGE) and the barrier breakdown mediated by HMGB1. In addition, gingerol inhibited the expression of CAMs and inhibited the attachment and migration of human neutrophils mediated by HMGB1. In addition, the inhibition of the activation of HMGB1-induced nuclear factor-κB (NF-κB) and extracellular regulated
본 명세서 실시예의 in vitro 및 in vivo 실험을 통하여 보인 바와 같이, 진저론은 HMGB1에 의해 매개되는 염증성 반응들을 억제하며, 이러한 HMGB1 signaling pathway의 억제를 통해 패혈증 및 패혈성 쇼크를 치료하는 효과가 있다. As shown in the in vitro and in vivo experiments of the examples of the present invention, gingerol suppresses inflammatory responses mediated by HMGB1 and is effective in treating sepsis and septic shock through inhibition of the HMGB1 signaling pathway .
따라서 본 발명은 진저론 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 예방 또는 치료용 약학적 조성물을 제공한다. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating septicemia or septic shock comprising gingerol or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 상기 패혈증(sepsis) 또는 패혈성 쇼크(septic shock)는 HMGB1에 의해 (High mobility group box 1)에 의해 매개되는 것이 특징으로, 상기“패혈증 또는 패혈성 쇼크의 예방 또는 치료”란, 폐혈증에 연관된 임상 증상 및 다기관 기능부전 증후군에 연관된 상태(예를 들어 다양한 정도의 열, 저독소혈증, 사성, 빈맥, 내피염, 심근경색증, 고도착란, 변화성 정신 상태, 혈관 허탈 및 기관 손상, 급성 호흡 곤란 증후군, 응고장애, 심부전증, 신부전증, 쇼크 및(또는) 혼수상태 등)의 전부 또는 일부 증상을 감소, 개선 또는 제거하는 것을 의미한다.The sepsis or septic shock of the present invention is characterized by being mediated by HMGB1 (High mobility group box 1). The term " preventing or treating septicemia or septic shock " (Including, for example, various degrees of heat, hypoxemia, spontaneous, tachycardia, endothelial dysfunction, myocardial infarction, elevated confusion, altered mental status, vasoconstriction and organ damage, acute Improvement or elimination of all or some of the symptoms of respiratory distress syndrome, coagulation disorders, heart failure, renal failure, shock and / or coma, and the like.
본 발명의 진저론은 생강으로부터 분리 정제하거나, 상업적으로 구입하여 사용하거나 또는 당업계에 공지된 화학적 합성법으로 제조할 수 있다. 예를 들어, 본 발명의 진저론은 생강으로부터 추출될 수 있다. 상기 화합물은 생강으로부터 유기용매 추출 및 크로마토그래피 등의 당업계에서 통상적으로 사용되는 공지의 방법에 의해 추출될 수 있다.The gingerol of the present invention can be separated and purified from ginger, used commercially or used, or can be prepared by chemical synthesis methods known in the art. For example, the ginger roots of the present invention can be extracted from ginger. The compound can be extracted by a known method commonly used in the art such as organic solvent extraction and chromatography from ginger.
본 발명에 따른 진저론은 그 자체 또는 약학적으로 허용 가능한 염의 형태로 사용될 수 있다. 상기에서 ‘약학적으로 허용되는’이란 생리학적으로 허용되고 인간에게 부여될 때, 통상적으로 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 비독성의 조성물을 말하여, 상기 염으로는 약학적으로 허용 가능한 유리산 (free acid)에 의하여 형성된 산 부가염이 바람직하다. 상기 유리산은 유기산과 무기산을 사용할 수 있다. 상기 유기산은 이에 제한되는 것은 아니나, 구연산, 초산, 젖산, 주석산, 말레인산, 푸마르산, 포름산, 프로피온산, 옥살산, 트리플로오로아세트산, 벤조산, 글루콘산, 메타술폰산, 글리콜산, 숙신산, 4-톨루엔술폰산, 글루탐산 및 아스파르트산을 포함한다. 또한, 상기 무기산은 이에 제한되는 것은 아니나 염산, 브롬산, 황산 및 인산을 포함한다.The gingerolone according to the present invention may be used as such or in the form of a pharmaceutically acceptable salt. The term " pharmaceutically acceptable " as used herein refers to a non-toxic composition that is physiologically acceptable and does not normally cause an allergic reaction or similar reaction when given to a human, Acid addition salts formed by free acids are preferred. The free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.
본 발명에 따른 약학적 조성물은 상기 진저론을 단독으로 함유하거나 약학적으로 허용되는 담체와 함께 적합한 형태로 제형화 될 수 있으며, 부형제 또는 희석제를 추가로 함유할 수 있다. 상기 담체로는 모든 종류의 용매, 분산매질, 수중유 또는 유중수 에멀젼, 수성 조성물, 리포좀, 마이크로비드 및 마이크로좀이 포함된다.The pharmaceutical composition according to the present invention may be formulated into a suitable form together with the above-mentioned gingerolone alone or together with a pharmaceutically acceptable carrier, and may further contain an excipient or a diluent. Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.
약학적으로 허용되는 담체로는 예컨대, 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 아울러, 경구투여용으로 사용되는 다양한 약물전달물질을 포함할 수 있다. 또한, 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코오스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르 브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸-또는 프로필-파라벤 및 클로로부탄올이 있다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현택제 등을 추가로 포함할 수 있다. 그 밖의 약학적으로 허용되는 담체 및 제제는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and preparations can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).
본 발명의 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들면, 경구 또는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다.The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다.The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.
경구 투여용 제제의 경우에 본 발명의 조성물은 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 등으로 당업계에 공지된 방법을 이용하여 제형화될 수 있다. 예를 들어, 경구용 제제는 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈,메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.
비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌(Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA,1995)에 기재되어 있다.In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa., 1995, which is a commonly known formulary for all pharmaceutical chemistries.
본 발명의 조성물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 바람직하게 본 발명의 약학적 조성물의 바람직한 전체 용량은 1일당 환자 체중 1㎏ 당 약 0.01㎍ 내지 10,000mg, 가장 바람직하게는 0.1㎍ 내지 500mg일 수 있다. 그러나 상기 약학적 조성물의 용량은 제제화 방법, 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율등 다양한 요인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 본 발명의 조성물의 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다. The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be from about 0.01 μg to about 10,000 mg, and most preferably from about 0.1 μg to 500 mg, per kilogram of patient body weight per day. However, the dosage of the pharmaceutical composition may be determined depending on various factors such as the formulation method, administration route and frequency of treatment, as well as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate, It will be possible to determine the appropriate effective dose of the composition of the present invention by those of ordinary skill in the art in view of this point. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
나아가, 본 발명의 약학적 조성물은 패혈증 또는 패혈성 쇼크의 예방 및 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수 있다.Furthermore, the pharmaceutical composition of the present invention can be administered in combination with a known compound having an effect of preventing and treating septicemia or septic shock.
아울러, 본 발명에 따른 상기 진저론 또는 이의 약학적으로 허용가능한 염은 패혈증 또는 패혈성 쇼크를 예방 또는 개선하기 위한 목적으로 식품 조성물의 형태로 제공될 수 있다. 본 발명의 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. 상기 유형의 식품 조성물은 당 업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.In addition, the gingerolone or a pharmaceutically acceptable salt thereof according to the present invention may be provided in the form of a food composition for the purpose of preventing or ameliorating septicemia or septic shock. The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.
예를 들면, 건강식품으로는 본 발명의 진저론 또는 이의 약학적으로 허용가능한 염 자체를 차, 주스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 패혈증 또는 패혈성 쇼크의 예방 및 개선효과가 있다고 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.For example, as a health food, the ginger rinse of the present invention or its pharmaceutically acceptable salt itself may be prepared in the form of tea, juice, and drink, and then taken for drinking, granulated, encapsulated and powdered. In addition, it can be prepared in the form of a composition by mixing with known substances or active ingredients known to be effective for preventing and improving septicemia or septic shock.
또한, 기능성 식품으로는 음료(알코올성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 진저론 또는 이의 약학적으로 허용 가능한 염을 첨가하여 제조할 수 있다.Functional foods also include beverages (including alcoholic beverages), fruits and processed foods (such as canned fruits, bottles, jams, maalmalade, etc.), fish, meat and processed foods such as ham, sausage, ), Breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, A preservative, a preservative, a preserved food, a frozen food, various kinds of seasonings (e.g., miso, soy sauce, sauce, etc.) by adding the ginger rosacea of the present invention or a pharmaceutically acceptable salt thereof.
본 발명의 식품 조성물 중 상기 진저론 또는 이의 약학적으로 허용 가능한 염의 바람직한 함유량으로는 이에 한정되지 않지만, 예를 들어, 최종적으로 제조된 식품 중 0.001 내지 30 중량% 일 수 있다. 바람직하게는 최종적으로 제조된 식품 중 0.01 내지 20 중량%일 수 있다.The preferred content of the gingerol or pharmaceutically acceptable salt thereof in the food composition of the present invention is not limited to, but may be, for example, 0.001 to 30% by weight of the finally prepared food. Preferably 0.01 to 20% by weight of the final produced food.
또한, 본 발명의 진저론 또는 이의 염을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.In order to use the gingerol or its salt of the present invention in the form of a food additive, it may be used in the form of a powder or a concentrated liquid.
본 발명의 진저론은 패혈증의 주요 매개인자인 HMGB1(High mobility group box 1)의 분비를 억제하고 상기 HMGB1과 관련된 전염증성 시그날링(proinflammatory signaling)을 억제하는 효과를 지니며, 실제로 in vivo 패혈증 동물 모델에서 패혈증의 치료효과가 현저하다.The ginger polysaccharide of the present invention suppresses secretion of HMGB1 (high mobility group box 1), which is a major mediator of sepsis, and inhibits proinflammatory signaling associated with HMGB1. In fact, in vivo sepsis The treatment effect of sepsis in the model is remarkable.
도 1A은 HUVEC 세포를 LPS(100ng/mL)로 16시간동안 자극한 후, 진저론(0.36mg/kg 또는 0.72mg/kg)을 6시간 처리하였을 때 HMGB1 분비량을 나타낸 것으로, 상기 HMGB1 분비량은 ELISA에 의해 측정되었다. (D: 0.2% DMSO vehicle control, *p < 0.05 versus LPS alone).
도 1B는 CLP를 수행한 수컷 C57BL/6 마우스들(n=5)에 CLP로부터 12시간 후, 말초혈관에 0.36 또는 0.72mg/kg 진저론을 정맥투여하고, CLP로부터 24시간 후에 상기 마우스를 안락사시켜 혈장 속 HMGB1 level을 측정한 결과를 나타낸 것으로, 상기 HMGB1 분비량은 ELISA에 의해 측정되었다. (D: 0.2% DMSO vehicle control, *p < 0.05 versus CLP alone).
도 1C은 HMGB1의 아세틸화에 대한 ZGR의 영향을 나타낸 것으로, HUVEC을 LPS 100ng/mL 로 처리하고 ZGR을 50μM 처리 또는 미처리 또는 SIRT1 억제제인 sirtinol 10mM을 처리한 상태로 6시간 배양 후 웨스턴 블롯으로 분석하였다. low 1은 HMGB1 단백질 수준을 항 acetyl-lysine(K)로 , low 2는 항 HMGB1으로, low 3은 배지에 16시간동안 배양한 후 항 HMGB1으로 측정한 결과이다.
도 1D는 HUVEC 에서의 SIRT1 발현에 대한 ZGR의 영향을 나타낸 것으로, HUVEC을 LPS 100ng/mL 로 처리하고 ZGR을 50μM 처리시 SIRT1의 시간당 발현량을 웨스턴블롯으로 측정한 것이다.
도 1E은 Confluent HUVEC 세포에 HMGB1(1μg/mL)을 16시간 처리하고 20μM or 50μM의 진저론을 처리하고 6시간 배양하였을 때, TLR-2, TLR-4 및 RAGE의 HMGB1 receptor들 발현을 나타낸 것으로, 상기 HMGB1 receptor들의 발현은 cell-based ELISA에 의하여 측정되었다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1 alone).
도 1F는 MTT assay 방법으로 세포 생존성(cell viability)에 대한 진저론의 영향을 측정한 결과를 나타낸다. (D: 0.2% DMSO vehicle control)
도 2A는 HUVEC 세포를 LPS (100ng/mL, 4 h)로 자극시킨 후, 표시된 각각의 농도로 진저론을 6시간 처리하였을 때 세포의 투과성을 평가한 결과를 나타내는 것으로, 상기 투과성은 HUVEC 단일층을 가로지른 Evans blue-결합된 알부민의 유출정도 측정에 의해 모니터링 되었다(D: 0.2% DMSO vehicle control, LPS: lipopolysaccharide, *p < 0.05 versus LPS).
도 2B은 HUVEC 세포를 HMGB1 (1μg/mL, 16 h)로 자극시킨 후, 표시된 각각의 농도로 진저론을 6시간 처리하였을 때 세포의 투과성을 평가한 결과를 나타내는 것으로, 상기 투과성은 HUVEC 단일층을 가로지른 Evans blue-결합된 알부민의 유출정도 측정에 의해 모니터링되었다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1).
도 2C은 HMGB1(2μg/mouse, i.v.)으로 마우스에 혈관투과(vascular permeability)를 유도한 후, 말초혈관에 진저론(0.36mg/kg 또는 0.72mg/kg)을 주입하여 이의 효과를 확인한 결과를 나타내는 것으로서, 복강 세척액(peritoneal washes)에 포함된 Evans blue 염료의 양을 측정하여 투과성을 평가하였다. (5마리의 마우스를 사용하였으며, 상기 투과된 염료의 양은 μg/mouse로 표현된다. D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1 alone).
도 2D은 HUVEC 세포를 HMGB1(1μg/mL, 16 h)으로 활성화시키고 서로 다른 농도의 진저론을 6시간동안 처리한 후, HMGB1에 의해 매개된 phospho-p38 발현에 대한 상기 각 화합물의 영향을 ELISA로 측정한 결과이다. (D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1 alone)
도 2E는 유리 커버슬립에서 자란 HUVEC 단층을 HMGB1로 1 시간 동안 자극한 다음, 6시간 동안 ZGR (25 또는 50μM)으로 처리하고 F-actin에 대해 염색한 결과이다. 화살표는 세포간극을 나타낸다.
도 3A는 HUVEC 세포를 HMGB1(1μg/mL)으로 16시간동안 자극하고 25μM 또는 50μM의 진저론을 6시간동안 처리한 후, HMGB1-매개 VCAM-1, ICAM-1 및 E-selectin의 발현 정도를 나타낸 것이다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1).
도 3B는 HUVEC 세포를 HMGB1 (1μg/mL)으로 16시간동안 자극하고 다양한 농도의 진저론으로 6시간동안 처리한 후, HUVEC monolayer에 대한 인간 호중구의 부착정도를 나타낸 것이다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1).
도 3C는 HUVEC 세포를 HMGB1 (1μg/mL)으로 16시간동안 자극하고 다양한 농도의 진저론(ZGR로 표기)으로 6시간동안 처리한 후, HUVEC monolayer에 대한 인간 호중구의 이동을 나타낸 것이다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1).
도 3D는 C57BL/6 수컷 마우스를 HMGB1(2μg per mouse, i.v.)으로 자극하고, 말초혈관에서 0.36mg/kg 또는 0.72mg/kg의 진저론을 주입하여, 상기 HMGB1에 의해 매개된 백혈구의 복막강으로의 이동(migration)을 분석한 결과를 나타낸다. (D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1).
도 3E는 HUVEC 세포를 HMGB1 (1μg/mL)으로 16시간동안 자극하고 25μM 또는 50μM 농도의 진저론으로 6시간동안 처리한 후, HUVEC monolayer에 대한 인간 호중구의 부착정도를 나타낸 것이다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1).
도 4A은 HUVECs 세포를 HMGB1 (1μg/mL)으로 16시간동안 자극하고 10μM, 25μM, 또는 50μM의 진저론을 6시간동안 처리한 후, HMGB1-매개 TNF-α의 생성 정도를 분석한 결과를 나타낸다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1)
도 4B는 HUVECs 세포를 HMGB1 (1μg/mL)으로 16시간동안 자극하고 10μM, 25μM, 또는 50μM의 진저론을 6시간동안 처리한 후, HMGB1-매개 IL-6의 생성 정도를 분석한 결과를 나타낸다(D: 0.2% DMSO vehicle control, *p < 0.05 versus HMGB1)
도 4C는 Confluent HUVECs 세포를 HMGB1 (1μg/mL)으로 16시간동안 자극하고 10μM, 25μM, 또는 50μM의 진저론을 6시간동안 처리한 후, HMGB1-매개 phospho-NF-κB p65 활성화 정도 및 전체 NF-κB p65를 분석한 결과를 나타낸다(D: 0.2% DMSO vehicle control, Active: phospho-NF-κB p65. Total: 전체 NF-κB p65, *p < 0.05 versus HMGB1)
도 4D는 Confluent HUVECs 세포를 HMGB1 (1μg/mL)으로 16시간동안 자극하고 10μM, 25μM, 또는 50μM의 진저론을 6시간동안 처리한 후, HMGB1-매개 phospho-ERK1/2 활성화 정도 및 전체 ERK1/2를 분석한 결과를 나타낸다(D: 0.2% DMSO vehicle control, Active: phospho-ERK1/2, Total: 전체 ERK1/2, *p < 0.05 versus HMGB1)
도 4E는 HUVEC 세포를 HMGB1 (1μg/mL)으로 자극하지 않거나 1시간동안 자극하였고, 6시간동안 ZGR (50μM) 로 처리하거나 전혀 처리하지 않았다. p65의 세포 내 위치를 IF 염색으로 검사하여 핵으로의 전이 여부를 Immunofluorescence 현미경 분석하였다. 이미지들은 세 번의 독립적 실험을 대표하는 것이다. (*p < 0.05 versus HMGB1)
도 5A는 수컷 C57BL/6 마우스들(n=20)에 CLP 후 12시간째 및 50시간째에 진저론(0.36mg/mouse, i.v. □) 또는 진저론(0.72mg/mouse, i.v. ■), 생강추출물(50mg/kg, I.v. △)을 정맥투여하고, CLP 후 132시간까지 6시간마다 실험동물의 생존을 모니터링한 결과를 나타낸다. Control CLP mice(●로 표시) 및 sham-operated mice (○로 표시)들에는 살균한 식염수가 투여되었다. CLP군에 대비한(versus) 전체 생존률을 조사하기 위하여 Kaplan-Meier survival analysis 방법이 사용되었다.
도 6A은 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.36mg/kg 또는 0.72mg/kg씩 진저론을 각각 정맥투여하고, CLP 후 96시간째에 안락사시켜 관찰한 폐조직의 조직병리학적 스코어(Histopathological score)를 나타낸다. (*p < 0.05 versus CLP)
도 6B은 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여하고, CLP 후 96시간째에 안락사시켜 수득한 폐조직을 H&E staining한 현미경사진(200x)이다; Sham group (grade 1); CLP group (grade 3); CLP + ZGR(grade 2); 이미지들은 세 번의 독립적 실험을 대표하는 것이다. (*p < 0.05 versus CLP)
도 6C는 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여한 후, 혈장 내 ALT 또는 AST 수준을 측정한 것이다. (*p < 0.05 versus CLP)
도 6D는 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여한 후, 혈장 내 크레아티닌 수준을 측정한 것이다. (*p < 0.05 versus CLP)
도 6E는 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여한 후, 혈장 내 BUN 수준을 측정한 것이다. (*p < 0.05 versus CLP)
도 6F는 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여한 후, 혈장 내 LDH 수준을 측정한 것이다. (*p < 0.05 versus CLP)
도 6G는 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여한 후, 혈장 내 T3 수준을 측정한 것이다. (*p < 0.05 versus CLP)
도 6H는 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여한 후, 혈장 내 T4 수준을 측정한 것이다. (*p < 0.05 versus CLP)
도 6I는 CLP 받은 수컷 C57BL/6 마우스(n=5)들에 CLP 후 12 및 50시간째에 0.72mg/kg씩 진저론을 정맥투여한 후, 혈장 내 TSH 수준을 측정한 것이다. (*p < 0.05 versus CLP) FIG. 1A shows the amount of HMGB1 secreted when HUVEC cells were stimulated with LPS (100 ng / mL) for 16 hours and treated with gingerol (0.36 mg / kg or 0.72 mg / kg) for 6 hours. Lt; / RTI > (D: 0.2% DMSO vehicle control, * p < 0.05 versus LPS alone).
FIG. 1B shows the results of intravenous administration of 0.36 or 0.72 mg / kg gingerrone to peripheral blood vessels after 12 hours from CLP in male C57BL / 6 mice (n = 5) undergoing CLP and after 24 hours from CLP, The level of HMGB1 secretion was measured by ELISA. (D: 0.2% DMSO vehicle control, * p < 0.05 versus CLP alone).
Fig. 1C shows the effect of ZGR on the acetylation of HMGB1. HUVEC was treated with 100 ng / mL of LPS and treated with 50 [mu] M of ZGR or treated with sirtinol 10 mM as the untreated or SIRT1 inhibitor for 6 hours and Western blot Respectively. low 1 is the result of measuring the HMGB1 protein level with the anti-acetyl-lysine (K), low 2 with the anti-HMGB1, and low 3 with the HMGB1 after culturing in the medium for 16 hours.
FIG. 1D shows the effect of ZGR on expression of SIRT1 in HUVEC. The expression level of SIRT1 per hour was measured by Western blotting when HUVEC was treated with
FIG. 1E shows expression of HMGB1 receptors in TLR-2, TLR-4, and RAGE when HMGB1 (1 μg / mL) was treated with Confluent HUVEC cells for 16 hours and 20 μM or 50 μM gingerol was treated for 6 hours , Expression of the HMGB1 receptors was measured by cell-based ELISA (D: 0.2% DMSO vehicle control, p < 0.05 versus HMGB1 alone).
FIG. 1F shows the results of measuring the effect of ginger on cell viability by MTT assay. (D: 0.2% DMSO vehicle control)
2A shows the results of evaluating the permeability of HUVEC cells after stimulation with HUVEC cells (100 ng / mL, 4 h) at the indicated concentrations for 6 hours, (D: 0.2% DMSO vehicle control, LPS: lipopolysaccharide, * p < 0.05 versus LPS) by measuring the efflux of Evans blue-conjugated albumin across the liver.
FIG. 2B shows the results of evaluating cell permeability when HUVEC cells were stimulated with HMGB1 (1 μg / mL, 16 h) and then treated for 6 hours with ginger at each of the indicated concentrations. The permeability was measured using a HUVEC monolayer (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1) by measuring the efflux of Evans blue-conjugated albumin across the membrane.
FIG. 2C shows the results obtained by injecting ginger (0.36 mg / kg or 0.72 mg / kg) into peripheral blood vessels after inducing vascular permeability in mice with HMGB1 (2 μg / mouse, iv) Permeability was assessed by measuring the amount of Evans blue dye contained in peritoneal washes. (5 mice were used, and the amount of the transmitted dye was expressed in [mu] g / mouse D: 0.2% DMSO vehicle control, p < 0.05 versus HMGB1 alone).
Figure 2D shows the effect of each compound on phospho-p38 expression mediated by HMGB1 after activation of HUVEC cells with HMGB1 (1 [mu] g / mL, 16 h) Respectively. (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1 alone)
FIG. 2E shows the result of stimulating the HUVEC monolayer grown in the glass cover slip with HMGB1 for 1 hour, then treating with ZGR (25 or 50 M) for 6 hours and staining for F-actin. The arrow indicates the cell gap.
FIG. 3A shows the expression levels of HMGB1-mediated VCAM-1, ICAM-1 and E-selectin after stimulation of HUVEC cells with HMGB1 (1 μg / mL) for 16 hours and 25 μM or 50 μM gingerrone treatment for 6 hours (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1).
Figure 3B shows the extent of human neutrophil adhesion to HUVEC monolayer after stimulation of HUVEC cells with HMGB1 (1 [mu] g / mL) for 16 hours and treatment with various concentrations of gingerolone for 6 hours (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1).
Figure 3C shows the migration of human neutrophils to HUVEC monolayer after stimulation of HUVEC cells with HMGB1 (1 [mu] g / mL) for 16 hours and treatment with various concentrations of gingerolone (denoted ZGR) for 6 hours (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1).
FIG. 3D shows the results of stimulation of C57BL / 6 male mice with HMGB1 (2 [mu] g per mouse, iv) and infusion of 0.36 mg / kg or 0.72 mg / kg gingerrone in peripheral blood vessels, The results are shown in Fig. (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1).
3E shows the degree of adhesion of human neutrophils to HUVEC monolayer after stimulation of HUVEC cells with HMGB1 (1 μg / mL) for 16 hours and treatment with 25 μM or 50 μM concentration of gingerolone for 6 hours (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1).
FIG. 4A shows the results of analysis of the degree of production of HMGB1-mediated TNF-α after stimulation of HUVECs cells with HMGB1 (1 μg / mL) for 16 hours and treatment with 10 μM, 25 μM, or 50 μM gingerol for 6 hours (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1)
FIG. 4B shows the results of analysis of the degree of production of HMGB1-mediated IL-6 after stimulation of HUVECs cells with HMGB1 (1 μg / mL) for 16 hours and treatment with 10 μM, 25 μM or 50 μM gingerol for 6 hours (D: 0.2% DMSO vehicle control, * p < 0.05 versus HMGB1)
Figure 4C shows that Confluent HUVECs cells were stimulated for 16 hours with HMGB1 (1 [mu] g / mL) and treated with 10, 25, or 50 [mu] M gingerol for 6 hours, followed by HMGB1-mediated phospho-NF- κB p65 (D: 0.2% DMSO vehicle control, Active: phospho-NF-κB p65. Total: total NF-κB p65, * p <0.05 versus HMGB1)
Figure 4D shows that Confluent HUVECs cells were stimulated with HMGB1 (1 [mu] g / mL) for 16 hours and then treated with 10 [mu] M, 25 [mu] M, or 50 [mu] M gingerol for 6 hours, followed by HMGB1-mediated phospho-ERK1 / 2 activation and total ERK1 / (D: 0.2% DMSO vehicle control, active: phospho-ERK1 / 2, Total: total ERK1 / 2, * p <0.05 versus HMGB1)
Figure 4E shows that HUVEC cells were not stimulated with HMGB1 (1 [mu] g / mL) or stimulated for 1 hour, treated with ZGR (50 [mu] M) for 6 hours or not at all. The intracellular location of p65 was examined by IF staining and immunohistochemical microscopic analysis of nuclear transfer was performed. The images represent three independent experiments. (* p < 0.05 versus HMGB1)
FIG. 5A shows the results of the administration of ginger (0.36 mg / mouse, iv) or ginger (0.72 mg / mouse, iv) at 12 hours and 50 hours after CLP in male C57BL / 6 mice (50 mg / kg, Iv < [Lambda] >) intravenously and monitoring the survival of the experimental animals every 6 hours up to 132 hours after CLP. Control CLP mice (marked with ●) and sham-operated mice (marked with ○) received sterile saline. The Kaplan-Meier survival analysis method was used to investigate the overall survival rate versus the CLP group.
FIG. 6A shows the results of intravenous administration of gingerrone at 0.36 mg / kg or 0.72 mg / kg at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP and 96 hours after CLP And the histopathological score of the lung tissue observed. (* p < 0.05 versus CLP)
FIG. 6B shows the results of pulmonary tissues obtained by intravenously administering gingerol at a dose of 0.72 mg / kg at 12 and 50 hours after CLP to male C57BL / 6 mice (n = 5) receiving CLP and euthanizing at 96 hours after CLP H & E stained microscope photograph (200x); Sham group (grade 1); CLP group (grade 3); CLP + ZGR (grade 2); The images represent three independent experiments. (* p < 0.05 versus CLP)
FIG. 6C is a graph showing ALT or AST levels in plasma after intravenous administration of gingerrone at a dose of 0.72 mg / kg at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP. (* p < 0.05 versus CLP)
FIG. 6D is a graph showing plasma creatinine levels measured after intravenous administration of gingerol at a dose of 0.72 mg / kg at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP. (* p < 0.05 versus CLP)
FIG. 6E shows plasma BUN levels after intravenous administration of ginger rats at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP at 0.72 mg / kg. (* p < 0.05 versus CLP)
FIG. 6F shows plasma LDH levels measured after intravenous administration of ginger rats at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP at 0.72 mg / kg. (* p < 0.05 versus CLP)
FIG. 6G shows plasma T3 levels measured after intravenous administration of gingerol at a dose of 0.72 mg / kg at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP. (* p < 0.05 versus CLP)
FIG. 6H shows plasma levels of T4 after intravenous administration of gingerrone at a dose of 0.72 mg / kg at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP. (* p < 0.05 versus CLP)
FIG. 6I shows plasma levels of TSH after intravenous administration of gingerrone at a dose of 0.72 mg / kg at 12 and 50 hours after CLP in male C57BL / 6 mice (n = 5) receiving CLP. (* p < 0.05 versus CLP)
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
<실험준비><Preparation of experiment>
1. 시약1. Reagents
진저론(Zingerone), 세균성 리포폴리사카라이드(LPS, 혈청형: 0111:B4, L5293), 에반스 블루(Evans blue), 크리스탈 바이올렛(crystal violet), 2-메르캅토에탄올(2-mercaptoethanol) 및 항생제(페니실린G 및 스트렙토마이신)를 Sigma (St.Louis,MO).로부터 수득하였다. 인간 재조합 HMGB1(Human recombinant high mobility group box 1 protein)은 Abnova (Taipei City, Taiwan)로부터 구입하였다. 소 혈청(Fetal bovine serum)) 및 Vybrant DiD는 Invitrogen (Carlsbad, CA)에서 구입 하였다.(2-mercaptoethanol), and antibiotics, such as zingerone, bacterial lipopolysaccharide (LPS, serotype: 0111: B4, L5293), Evans blue, crystal violet, (Penicillin G and streptomycin) were obtained from Sigma (St. Louis, Mo.). Human recombinant high
2. 세포 배양2. Cell culture
인간 탯줄 정맥 내피세포(Primary human umbilical vein endothelial cells, HUVECs)는 Cambrex Bio Science(Charles City, IA, USA)로부터 수득하였고, Bae et al., 2014;의 연구에서 묘사된 바와 같이 유지되었다(maintained). 간략하게, growth supplements(Cambrex Bio Science)가 첨가된 EBM-2 basal media 용액에서 37℃, 5% CO2 조건으로, 세포들이 포화(confluency)되도록 배양하였다. 모든 실험은 3-5계대(passage) 배양된 HUVEC 세포를 사용하여 수행되었다. 인간 호중구(neutrophil)는 다섯명의 건강한 지원자로부터 정맥천자(venipuncture)에 의해 수득된 15 mL의 전혈(whole blood)로부터 분리되었고, Hofbauer R et al., 1998의 연구에서 묘사된 바와 같이 유지되었다.Human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex Bio Science (Charles City, IA, USA) and maintained as described in Bae et al., 2014; . Briefly, cells were incubated in EBM-2 basal media solution supplemented with growth supplements (Cambrex Bio Science) at 37 ° C and 5% CO 2 for confluency. All experiments were performed using 3-5 passaged HUVEC cells. Human neutrophil was isolated from 15 mL of whole blood obtained by venipuncture from five healthy volunteers and maintained as described in Hofbauer R et al., 1998.
3. 실험동물 및 사육3. Experimental animals and breeding
몸무게가 27g인 6 내지 7주령 C57BL/6 수컷 마우스들을 orient Bio Co.(성남, 대한민국)로부터 구입하였고 12개월의 적응기간(acclimatization period) 후에 본 연구에 이용하였다. 실험동물들은 polycarbonate cage 당 5마리씩, 20-25℃의 온도, 40%-45%의 습도 및 12-22 시간 주기의 낮/밤 순환 조건에서 사육되었다. 상기 적응기간 동안, 실험동물들은 일반 설치류 펠렛 사료(rodent pellet diet) 및 물을 자유식이(ad libitum)하였다. 모든 동물은 Kyungpook National University에 의해 발행된‘for the Care and Use of Laboratory Animals’에 따라 처리되었다(IRB No. KNU 2016-54)6-7 week old C57BL / 6 male mice weighing 27 g were purchased from orient Bio Co. (Seongnam, Korea) and used for this study after an acclimatization period of 12 months. The animals were housed in polycarbonate cages at a temperature of 20-25 ° C, humidity of 40% -45% and day / night circulation of 12-22 hours. During the adaptation period, the experimental animals ad libitum the rodent pellet diet and water. All animals were treated according to the 'for the Care and Use of Laboratory Animals' issued by Kyungpook National University (IRB No. KNU 2016-54)
4. CLP(Cecal Ligation and Puncture)4. CLP (Cecal Ligation and Puncture)
패혈증을 유도하기 위해, 수컷 마우스들은 졸레틸(틸레타민과 졸라제팜, 1 : 1 혼합물, 30 mg/kg)과 럼푼(자일라진, 10mg/kg)로 마취되었다. 간략하게, 2cm의 정중절개(midline incision)를 수행하여 맹장 및 인접한 소장을 노출시켰다. 그 후 맹장 끝부분(cecal tip)으로부터 5.0mm 떨어진 부분을 3.0-비단봉합사(silk suture)로 단단히 묶고, 높은 수준의 패혈증을 유발하기 위하여 22-게이지의 바늘로 맹장에 1회 구멍을 냈다. 그 후, 상기 구멍 부위로부터 배설물을 압출시키기 위하여 맹장을 부드럽게 짜내었고(squeezed), 상기 맹장을 다시 복막강(peritoneal cavity)으로 복귀시켰다. 마지막으로 개복 부위(laparotomy site)를 4.0-silk로 봉합하였다. sham control 군의 실험동물은 맹장을 노출시킨 후 봉합(ligation) 또는 구멍을 내는 과정 없이 복강으로 복귀시켰다. 상기 실험 프로토콜은 실험 전, the Animal Care Committee at Kyungpook National University(IRB No. KNU 2016-54)에 의해 승인 받았다.To induce sepsis, male mice were anesthetized with zoletile (tiletamine and zolazepam, 1: 1 mixture, 30 mg / kg) and rumun (xylazine, 10 mg / kg). Briefly, a 2 cm midline incision was performed to expose the cecum and adjacent small intestine. A 5.0-mm section from the cecal tip was then tightly bound with a 3.0-silk suture and punctured once with a 22-gauge needle to induce a high level of sepsis. The cecum was then gently squeezed to extrude the excrement from the hole and the cecum was returned to the peritoneal cavity. Finally, the laparotomy site was closed with 4.0-silk. The animals in the sham control group were exposed to the abdominal cavity without exposing the cecum and ligation or puncturing. Prior to the experiment, the experimental protocol was approved by the Animal Care Committee at Kyungpook National University (IRB No. KNU 2016-54).
5.5. HMGB1 측정을 위한 경쟁적 ELISA(Competitive enzyme-linked immunosorbent assay)Competitive enzyme-linked immunosorbent assay (ELISA)
경쟁적 ELISA 방법을 이용하여 HMGB1의 농도를 조사하였다. HUVEC 단일층(HUVEC monolayer)은 LPS(100ng/mL)로 16시간 동안 처리되었으며, 그 후 진저론으로 6시간동안 처리되었다. 그 다음에, HMGB1의 농도를 조사하기 위하여 세포배양액을 수집하였다. ELISA를 수행하기 위하여, 96-well flat plastic microtiter plates(Corning Inc., Corning, NY)를 HMGB1 단백질과 함께 4℃에서 오버나잇(overnight)하여 코팅하였으며, 이때 0.02% sodium azide가 포함된 20mM carbonate-bicarbonate buffer(pH 9.6)를 사용하였다. 상기 plate를 0.05% Tween 20이 포함된 PBS 용액(PBS-T)으로 3회 세척한 뒤, 4℃에서 보관하였다. 상기 수집된 세포배양액은 동결건조(Lyophilization)시킨 후, 이를 anti-HMGB1 antibody(PBS-T에 1:1000로 희석됨, Abnova, Taipei City, Taiwan)와 함께 96-well plastic round microtiter plate에서 37℃, 90분 동안 사전-배양(pre-incubate)하였고, 이를 상기 HMGB1으로 코팅된 플레이트에 옮긴 후, 30분동안 실온에서 배양하였다. 그리고나서 상기 플레이트를 PBS-T 용액으로 3회 세척하고, peroxidase-conjugated anti-rabbit IgG antibody(PBS-T에 1:2000로 희석됨, Amersham Pharmacia Biotech)와 함께 실온에서 90분동안 배양하였으며, 다시 PBS-T 용액으로 3회 세척하고, 200㎕의 기질용액(100μg/mL o-phenylenediamine 및 0.003% H2O2)을 첨가하여 암실에서 상온으로 60분 동안 배양하였다. 8N H2SO4를 50㎕ 처리하여 반응을 종료시킨 후, 490nm에서 흡광도를 측정하였다. The concentration of HMGB1 was investigated using a competitive ELISA method. HUVEC monolayers were treated with LPS (100 ng / mL) for 16 h and then treated with gingerol for 6 h. Then, cell culture medium was collected to examine the concentration of HMGB1. To perform the ELISA, 96-well flat plastic microtiter plates (Corning Inc., Corning, NY) were coated with HMGB1 protein at 4 ° C overnight, in which 20 mM carbonate- bicarbonate buffer (pH 9.6) was used. The plate was washed three times with PBS solution (PBS-T) containing 0.05
6. 면역침전 및 웨스턴 블롯(Western blot)6. Immunoprecipitation and Western blotting
세포를 Pierce immunoprecipitation (IP) Lysis Buffer (Thermo Fisher Scientific) 200 ㎕에 녹인 후 4 ℃에서 15,000 g으로 15분간 원심분리 하였다. 단백질 추출 후, 200-300 ㎍의 총 세포 단백질을 4 ℃에서 1 시간 동안 Protein G Sepharose 4 Fast Flow (GE Healthcare Life Sciences, Buckinghamshire, UK)로 미리 털고 잠시 원심분리 하였다. 미리 씻어 낸 세포 용 해물을 항 HMGB1 (Santa Cruz Biotechnology)과 함께 4 ℃에서 밤새 항온 배양 한 후 Protein G Agarose로 4 시간 배양했다. 원심분리 후, 세파로스 비드(Sepharose bead)를 PBS로 세척하고 웨스턴 블롯으로 분석 하였다.Cells were dissolved in 200 μl of Pierce immunoprecipitation (IP) lysis buffer (Thermo Fisher Scientific) and centrifuged at 15,000 g for 15 min at 4 ° C. After protein extraction, 200-300 [mu] g of total cellular protein was prewashed with
웨스턴 블롯을 위해, 세포를 PBS로 헹구고 0.5% SDS, 1 % NP-40, 1 % 나트륨 데옥시콜레이트, 150mM NaCl, 50mM Tris-Cl (pH 7.5) 및 프로테아제 억제제. 브래드포드 (Bradford) 분석을 사용하여 각 샘플의 단백질 농도를 결정하고, ELISA 플레이트 판독기를 사용하여 595 nm에서의 혼합물의 흡광도를 측정 하였다. 상층 액에서 HMGB1의 분비를 검출하기 위해, 배양 배지의 샘플을 간단히 원심 분리하여 세포 파편을 제거 하였다. 동일한 양의 각 시료를 2x loading dye와 혼합하고 95 ℃에서 5 분간 끓여 주었다. 배양 배지 및 전체 세포 용해물 샘플을 관심있는 단백질의 크기에 따라 상이한 퍼센트의 폴리아크릴아미드 겔에서 전기영동 하였다. 겔을 2시간 동안 20 mA에서 반건조 전기영동 이동을 통해 PVDF막으로 옮겼다. PVDF막을 5% 소 혈청 알부민(BSA)에서 실온에서 2 시간 동안 차단시켰다. 그런 다음 멤브레인을 4℃에서 밤새 5% BSA를 함유하는 Tris-완충 식염수 / Tween 20 (TBS-T)에서 1 : 500으로 희석한 1차 항체와 함께 배양한 다음 2차 항체 (1 : 5000 희석 TBS-T 1 % BSA 함유)에 실온에서 1시간동안 처리하였다. TBS-T 용액으로 3회 세척한 후, 멤브레인을 ECL 검출 시약 (Amersham, Piscataway, NJ)과 함께 배양하고 Xomat AR 필름 (Eastman Kodak, Rochester, NY)에 노출시켰다. 스캐닝 농도계는 Image Master VDS (Pharmacia Biotech Inc., San Francisco, CA)를 사용하여 수행 하였다.For Western blot, cells were rinsed with PBS and incubated with 0.5% SDS, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl (pH 7.5) and protease inhibitor. The protein concentration of each sample was determined using Bradford analysis and the absorbance of the mixture at 595 nm was measured using an ELISA plate reader. To detect the secretion of HMGB1 in the supernatant, a sample of the culture medium was briefly centrifuged to remove cell debris. The same amount of each sample was mixed with a 2x loading dye and boiled at 95 ° C for 5 minutes. The culture medium and whole cell lysate samples were electrophoresed on different percentages of polyacrylamide gels depending on the size of the protein of interest. The gel was transferred to the PVDF membrane through semi-dry electrophoresis at 20 mA for 2 hours. The PVDF membrane was blocked with 5% bovine serum albumin (BSA) at room temperature for 2 hours. Membranes were then incubated with primary antibody diluted 1: 500 in Tris-buffered saline / Tween 20 (TBS-T) containing 5% BSA overnight at 4 ° C and then incubated with secondary antibody (1: 5000 dilution TBS -
7. 세포 생존 에세이(Cell viability assay)7. Cell viability assay
세포 생존의 지표로서 MTT assay 방법을 이용하였다. 세포들은 96-well plates에서 5 ×103 cells/well의 밀도로 배양되었다. 24시간 후에, 세포들을 새 배지(fresh medium)로 세척하였으며, 진저론을 처리하였다. 48시간의 배양 후에, 세포를 세척하고 100㎕의 MTT(1mg/mL)를 첨가하여 4시간 배양하였다. 마지막으로 DMSO(dimethyl sulfoxide) 150㎕를 첨가하여 를 용해시키고, formazan salt의 양을 microplate reader(Tecan Austria GmbH, Austria)를 사용하여 540nm에서 OD(optical density)를 측정함으로서 조사하였다. MTT assay was used as an indicator of cell survival. Cells were cultured at a density of 5 × 10 3 cells / well in 96-well plates. After 24 hours, the cells were washed with fresh medium and treated with gingerolone. After 48 hours of culture, the cells were washed and 100 의 of MTT (1 mg / mL) was added and incubated for 4 hours. Finally, 150 μl of DMSO (dimethyl sulfoxide) was added to dissolve and the amount of formazan salt was examined by measuring OD (optical density) at 540 nm using a microplate reader (Tecan Austria GmbH, Austria).
8. 8. in vitro in vitro 투과성 에세이 (Permeability assay)Permeability assay
Qureshi et al., 2008의 연구에서 기술된 바와 같은 modified 2-compartment chamber model을 이용하여, 기능적 HUVEC 세포 단일층(functional HUVEC monolayer)을 가로지른 Evans blue-결합된 알부민의 유출(flux)을 분광광도 측정(spectrophotometric measurement)하는 방법에 의해 투과성을 정량하였다. 간략하게, HUVEC 세포들은 3μm pore size, 12mm 직경의 transwell 에 5 X104/well 로 분주되었고, 3일 동안 배양되었다. 이렇게 생성된 HUVEC 융합 단층(Confluent monolayers)에 LPS (100 ng/mL, 4h) 또는 HMGB1(1㎍/mL, 16h)을 처리하고, 그리고나서 진저론을 6시간동안 처리하였다. 이어서, 트랜스웰 삽입물을 PBS (pH 7.4)로 세척하고, 0.5mL의 Evans blue(0.67mg/mL) 및 4% BSA를 함유하는 성장 배지를 첨가 하였다. 신선한 성장 배지를 하부 chamber에 첨가하고, 상부 chamber의 배지를 Evans blue/ BSA로 대체 하였다. 10분 후, 하부 chamber 내의 광학밀도를 650nm에서 측정 하였다.Using a modified 2-compartment chamber model as described in the Qureshi et al., 2008 study, the flux of Evans blue-bound albumin across a functional HUVEC monolayer was measured spectrophotometrically The permeability was quantified by a spectrophotometric measurement method. Briefly, HUVEC cells were plated at 5 × 10 4 / well in 3 μm pore size, 12 mm diameter transwells and cultured for 3 days. Confluent monolayers were treated with LPS (100 ng / mL, 4 h) or HMGB1 (1 μg / mL, 16 h) and then treated with gingerolin for 6 h. The transwell inserts were then washed with PBS (pH 7.4) and growth medium containing 0.5 mL of Evans blue (0.67 mg / mL) and 4% BSA was added. Fresh growth medium was added to the lower chamber, and the upper chamber medium was replaced with Evans blue / BSA. After 10 minutes, the optical density in the lower chamber was measured at 650 nm.
9. 세포-세포 부착 어세이(Cell-Cell adhesion assay)9. Cell-Cell adhesion assay (Cell-Cell adhesion assay)
정제된 인간 호중구(1.5 ×106/mL, 200㎕/well)세포들을 Vybrant DiD dye로 표지하고, 세척 및 자극된 HUVEC 세포들에 상기 호중구 세포들을 첨가하였다. 상기 자극된 HUVEC 세포는, HUVEC 세포 단층을 HMGB1 (1 ㎍/mL)으로 16시간동안 처리하고, 6시간동안 진저론을 처리한 것이다. Purified human neutrophils (1.5 × 10 6 / mL, 200 μl / well) were labeled with Vybrant DiD dye and the neutrophils were added to the washed and stimulated HUVEC cells. The stimulated HUVEC cells were treated with HMGB1 (1 [mu] g / mL) for 16 hours and treated with ginger rinse for 6 hours.
10. In vitro 10. In vitro 이동성 에세이(migration assay)Migration assay
세포 이동성 에세이는 8μm pore size filter를 포함하는 6.5mm직경의 transwell plate에서 수행되었다. HUVEC 세포(6 X 104)들을 3일동안 배양하여, 융합된 내피세포 단일층을 수득하였다. 상기 세포 단층을 HMGB1(1㎍/mL)으로 16시간 처리하고 0 내지 20μM의 각 평가대상 화합물(비세닌-2 또는 스콜리모사이드)을 6시간동안 처리한 후, transwell plate의 상부(upper compartment)에 인간 호중구를 첨가하였다. 그 후 상기 Transwell plate 는 37°5% CO2 조건에서 2시간동안 배양되었다. 그 후 upper chamber의 세포들은 흡인되었으며(aspirated), 필터의 윗부분에 위치한 비이동성 세포들은 면봉을 이용하여 제거되었다. 필터의 아랫부분에 위치한 인간 호중구 세포들은 8% glutaraldehyde로 고정된 후, 0.25% crystal violet을 포함하는 20% methanol(w/v)을 첨가하여 염색되었다. 각각의 실험은 2배수 웰(duplicate well)에서 반복되었으며, 무작위로 선택된 9개의 high power microscopic field(HPF; 200×)들에서 이동성 세포의 개수가 카운팅(counting)되었다. 이러한 결과는 이동 지표(migration index)로서 나타내어진다.The cell mobility assay was performed on a 6.5 mm diameter transwell plate containing an 8 μm pore size filter. HUVEC cells (6 × 10 4 ) were cultured for 3 days to obtain a monolayer of fused endothelial cells. The cell monolayer was treated with HMGB1 (1 μg / mL) for 16 hours, treated with 0 to 20 μM of each compound to be evaluated (bisenin-2 or squalomeside) for 6 hours, Human neutrophils were added. The Transwell plate was then incubated for 2 hours at 37 ° C in 5% CO 2 . The cells in the upper chamber were then aspirated and non-migrating cells located at the top of the filter were removed using a cotton swab. Human neutrophils at the bottom of the filter were fixed with 8% glutaraldehyde and stained with 20% methanol (w / v) containing 0.25% crystal violet. Each experiment was repeated in duplicate wells and the number of migrating cells was counted in nine randomly selected high power microscopic fields (HPF; 200x). These results are expressed as migration index.
11.11. In vivo In vivo 투과성 및 백혈구 이동성 에세이Permeability and leukocyte mobility essay
in vivo 실험을 위하여, 수컷 마우스들은 small rodent gas anesthesia machine(RC2, Vetequip, Pleasanton, CA)을 통하여 주입된 2% isoflurane(Forane, JW Pharmaceutical, South Korea)이 포함된 산소로 마취되었고, 최초에는 breathing chamber에서 마취하고 그 후에는 facemask를 사용하여 실험과정동안 자연스럽게 호흡할 수 있도록 하였다. 마우스들에 HMGB1(2㎍/mouse)을 정맥주사(i.v.)하여 16시간동안 전처리하고, 진저론 (0.36 또는 0.72 mg / kg, i.v.)으로 처리 하였다. For in vivo experiments, male mice were anesthetized with oxygen containing 2% isoflurane (Forane, JW Pharmaceutical, South Korea) injected via a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton, CA) The chamber was anesthetized and then a facemask was used to allow the patient to breathe naturally during the course of the experiment. Mice were pretreated with HMGB1 (2 μg / mouse) intravenously (iv) for 16 h and treated with gingerol (0.36 or 0.72 mg / kg, iv).
in vivo 투과성 에세이를 위하여, 1% Evans blue dye solution(in normal saline)을 각 마우스에 정맥주사하였다. 30분 후에, 각 마우스들을 희생시켰으며, 5mL의 일반 식염수로 복막강을 세척하여 복막삼출물(peritoneal exudate)들을 수집하고 10분동안 200Xg으로 원심분리하였다. 상등액의 흡광도는 650nm에서 측정되었다. Bae JS et al., 2012의 연구에서 기술된 바와 같이, 혈관 투과성은 Evans blue dye의 표준 곡선을 이용하여 측정된 복막강(peritoneal cavity)으로 누설(leak)된 염료의 양(μg of dye/mouse)으로서 나타내었다. For in vivo permeability assay, 1% Evans blue dye solution (in normal saline) was intravenously injected into each mouse. After 30 minutes, each mouse was sacrificed and peritoneal exudates were collected by washing the bicomponent flasks with 5 mL of normal saline and centrifuged at 200 xg for 10 minutes. The absorbance of the supernatant was measured at 650 nm. As described in Bae JS et al., 2012, vascular permeability is the amount of dye (μg of dye / mouse) leaked into the peritoneal cavity measured using the standard curve of Evans blue dye ).
백혈구 이동을 평가하기 위하여, 상기와 같이 각 평가대상 화합물을 처리하고 6시간 후에 마우스들을 희생하였고, 복막강을 5 mL의 일반 식염수로 세척하였다. 20㎕의 상기 복막액(peritoneal fluid) 샘플을 0.38 mL Turk’solution(0.01% crystal violet in 3% acetic acid)과 혼합하고, 광학현미경을 이용하여 백혈구의 수를 카운팅(counting)하였다.To assess leukocyte migration, each subject compound was treated as described above, and after 6 hours mice were sacrificed and the bicuspid steal was washed with 5 mL of normal saline. Twenty microliters of the peritoneal fluid sample was mixed with 0.38 mL of Turk's solution (0.01% crystal violet in 3% acetic acid) and the number of white blood cells was counted using an optical microscope.
12. 세포 부착 분자 (CAMs)와 HMGB1 수용체의 발현12. Expression of cell adhesion molecules (CAMs) and HMGB1 receptor
VCAM-1, ICAM-1 및 E-selection의 발현은 whole-cell ELISA를 통해 확인하였다. 간단히 말하면, HUVEC의 융합 단일층을 HMGB1(1㎍/mL)로 16시간 (VCAM-1 및 ICAM-1) 또는 22시간 (E-selectin) 처리하고, 진저론으로 처리하고, 1% paraformaldehyde로 고정시켰다. 3회 세척한 후 마우스 항-인간 단일클론항체 (VCAM-1, ICAM-1 및 E-selectin, Temecula, CA, 각각 1:50)를 첨가하고 샘플을 1시간동안 배양하였다(37℃, 5%CO2). 그 다음, 세포를 세척하고 peroxidase-conjugated 항 마우스 IgG 항체 (Sigma, St. Louis, MO)로 1 시간 동안 처리하고 3회 세척한 다음 o-phenylenediamine substrate(Sigma, St. Louis, MO)로 처리 하였다. Expression of VCAM-1, ICAM-1 and E-selection was confirmed by whole-cell ELISA. Briefly, fusion monolayers of HUVEC were treated with HMGB1 (1 μg / mL) for 16 hours (VCAM-1 and ICAM-1) or 22 hours (E-selectin), treated with gingerolone and fixed with 1% paraformaldehyde . After washing three times, mouse anti-human monoclonal antibodies (VCAM-1, ICAM-1 and E-selectin, Temecula, CA, 1:50 each) were added and samples were incubated for 1 hour CO2). Cells were then washed and treated with peroxidase-conjugated anti-mouse IgG antibody (Sigma, St. Louis, MO) for 1 h, washed three times and then treated with o-phenylenediamine substrate (Sigma, St. Louis, MO) .
상기와 같은 실험과정은 TLR2, TLR4 및 RAGE와 같은 HMGB1 리셉터들의 세포표면 발현을 모니터링하기 위해 이용되었으며, 이때 Santa Cruz Biotechnology Inc.(Santa Cruz, CA)로부터 수득한 상기 receptor별 특수항체(각각 A-9, H-80 및 A-9)들을 사용하였다.The experimental procedure described above was used to monitor the cell surface expression of HMGB1 receptors such as TLR2, TLR4 and RAGE, wherein the specific antibodies of the receptor (obtained from Santa Cruz Biotechnology Inc., Santa Cruz, Calif. 9, H-80 and A-9).
13. 인산화된 p38 MAPK, NF-κB, TNF-α, ERK 1/2 및 IL-6의 측정을 위한 ELISA13. ELISA for determination of phosphorylated p38 MAPK, NF-kB, TNF-a,
인산화된 p38 MAPK(phosphorylated p38 mitogen-activated protein kinase)의 발현은 Cell Signaling Technology(Danvers, MA, USA)사의 상용화 ELISA kit를 제조사의 프로토콜에 따라 사용하여 정량하였다. 핵 용해물(nuclear lysates)에서 전체(total) 및 인산화된 p65 NF-κB의 활성은 Cell Signaling Technology(Danvers, MA)사로 부터의 ELISA kit(Catalog NO. #7174, #7173)를, 전체(total) 및 인산화된 ERK 1/2(extracellular regulated kinase 1/2)의 활성은 R&D Systems(Minneapolis, MN, USA)사의 ELISA kit를 사용하여 조사하였다. 세포 배양액의 상층액에서 IL-6 및 TNF-α의 농도는 R&D Systems(Minneapolis, MN, USA)의 ELISA kit를 이용하여 조사하였다. ELISA plate reader (Tecan, Austria GmbH, Austria)를 이용하여 값을 측정하였다.The expression of phosphorylated p38 mitogen-activated protein kinase (phosphorylated p38) was quantitated using a commercial ELISA kit from Cell Signaling Technology (Danvers, MA, USA) according to the manufacturer's protocol. The activity of total and phosphorylated p65 NF-κB in nuclear lysates was determined by ELISA kit (Catalog NO. # 7174, # 7173) from Cell Signaling Technology (Danvers, MA) ) And phosphorylated
14. 헤마톡실린 & 에오신 염색 및 병리조직학적 시험14. Hematoxylin & eosin staining and histopathological examination
5마리의 수컷 C57BL/6 마우스들에 CLP 를 수행하고 각각 12시간 및 50시간 후에, 진저론 (0.36 또는 0.72mg/kg, i.v.)을 투여 받았다. 마우스들은 CLP 후 96시간째에 안락사되었다. 상기 마우스들에서 폐조직의 표현형 변화를 분석하기 위하여 각 마우스로부터 폐 샘플들을 적출하였고, 잔류 혈액을 제거하기 위하여 PBS(pH 7.4)로 3회 세척한 후 4% formaldehyde solution(in PBS (pH 7.4), Junsei, Tokyo, Japan)으로 4℃에서 20시간동안 고정하였다. 상기 고정 후, 각 샘플들은 일련의 증가되는 농도의 에탄올 수용액(ethanol series)으로 탈수되었고, 파라핀 포매된 후, 4μm두께로 절편화하여 슬라이드(slide)에 올려놓았다. 상기 슬라이드들은 60℃ 오븐에서 탈파라핀화(deparaffinize)되었고, 재수화(rehydrate)된 후, hematoxylin(Sigma)으로 염색되었다. 과도한 염색을 제거하기 위하여 각 슬라이드들을 0.3% acid alcohol에 3회 급속침지(quick-dip)시켰으며, eosin(Sigma)으로 대비염색하였다. 그리고나서 일련의 증가되는 농도의 에탄올 수용액(ethanol series) 및 자일렌(xylene)을 이용하여 과도한 염색을 제거하고, 커버슬립(coverslip)을 덮었다. 폐 조직구조(pulmonary architecture), 조직 부종(tissue edema) 및 염증성 세포의 침윤(infiltration of inflammatory cell)을 평가하기 위한 폐 표본에 대한 광학현미경분석은, Ozdulger A et al., 2003에 기술된 바와 같이 블라인드 관찰자(blinded observation)에 의해 수행되었다. 상기 관찰 결과는 하기의 4등급으로 분류되었다: 등급 1(grade 1)은 정상 조직병리(normal histopathology)를 나타내고, 등급 2(grade 2)는 소량의 호중구 백혈구 침윤을 나타내며, 등급 3(grade 3)은 중간정도의 호중구 백혈구 침윤, 혈관주위 부종(perivascular edema) 형성 및 폐 조직구조의 부분적 파괴를 나타내고, 등급 4(grade 4)는 밀도 높은 호중구 백혈구 침윤, 농양(abscess) 형성 및 완전한 폐 조직구조 파괴를 포함하는 의미이다.Five male C57BL / 6 mice were treated with ginger ronin (0.36 or 0.72 mg / kg, i.v.) after 12 h and 50 h, respectively, following CLP. The mice were euthanized at 96 hours after CLP. In order to analyze the phenotypic changes of lung tissue in the mice, lung samples were extracted from each mouse, washed with PBS (pH 7.4) three times to remove residual blood, and then resuspended in 4% formaldehyde solution (pH 7.4) , Junsei, Tokyo, Japan) at 4 ° C for 20 hours. After the fixation, each sample was dehydrated with a series of increasing concentrations of ethanol series, paraffin-embedded, cut into 4 μm thick slices and placed on a slide. The slides were deparaffinized in a 60 ° C oven, rehydrated and stained with hematoxylin (Sigma). To eliminate excessive staining, each slide was rapidly dipped into 0.3% acid alcohol three times, and contrasted with eosin (Sigma). Excess dyeing was then removed with a series of increasing concentrations of ethanol series and xylene, and coverslips were covered. Optical microscopy analysis of lung specimens for evaluating pulmonary architecture, tissue edema and infiltration of inflammatory cells was performed as described in Ozdulger A et al., 2003 Blinded < / RTI > observation. These observations were classified into the following four grades:
15. 면역 형광 염색 실험15. Immunofluorescent staining experiment
HUVEC는 10% FBS를 함유하는 완전 배지에서 0.05% Poly-L-Lysine으로 코팅 된 유리 커버 슬립 상에 화체시키고 48시간동안 유지시켰다. 그 후 세포를 6 시간 진저론(25 또는 50μM)처리한 것에 관계없이 16시간 동안 HMGB1(1㎍/mL)로 자극하였다. 세포골격 염색을 위해 실온에서 15분간 PBS(v/v)에서 4% 포름알데히드로 고정시키고, PBS 중 0.05% Triton X-100에서 15 분간 투과성으로 하고, 블로킹 완충액 (5 % BSA in PBS) 4°에서 하룻밤동안 유지시켰다. 그런 다음 세포를 하루동안 4℃에서 F-actin이 표지 된 fluorescein phalloidin(F 432; 분자 프로브, Invitrogen) 또는 1차 토끼 단일 클론 NF-κB p65 항체 및 항-토끼 alexa 488과 함께 배양하였다. 핵을 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)로 대조 염색하고 630x 배율의 confocal 현미경(TCS-Sp5, Leica microsystem, Germany)으로 관찰하였다.HUVEC was stained on a glass cover slip coated with 0.05% Poly-L-Lysine in complete medium containing 10% FBS and kept for 48 hours. The cells were then stimulated with HMGB1 (1 [mu] g / mL) for 16 hours, regardless of whether the cells were treated for 6 hours with gingerolone (25 or 50 [mu] M). The cells were fixed with 4% formaldehyde in PBS (v / v) for 15 min at room temperature, permeabilized for 15 min with 0.05% Triton X-100 in PBS, blocked with blocking buffer (5% BSA in PBS) ≪ / RTI > overnight. Cells were then incubated with F-actin labeled fluorescein phalloidin (F 432; molecular probe, Invitrogen) or primary rabbit monoclonal NF-κB p65 antibody and anti-rabbit alexa 488 at 4 ° C for one day. Nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed with confocal microscopy (TCS-Sp5, Leica microsystem, Germany) at 630x magnification.
16. 장기 손상 마커 및 갑상선 호르몬 측정16. Measurement of organ damage marker and thyroid hormone
상용 분석 키트(Pointe Scientific, Linclon Park, MI)를 사용하여 Aspartate transaminase(AST), alanine transaminase(ALT), 혈액 요소 질소(BUN) 및 크레아티닌의 혈장 수준을 측정 하였다. Triiodothyronine(T3), thyroxine(T4) 와 갑상선 자극 호르몬(TSH) (T3 and T4, Calbiotech, Spring Valley, CA) or (TSH, LSBio, Seattle, WA) 또한 상용 분석 키트를 사용하여 측정하였다.Plasma levels of aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN) and creatinine were measured using a commercial assay kit (Pointe Scientific, Linclon Park, MI). Triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) (T3 and T4, Calbiotech, Spring Valley, CA) or (TSH, LSBio, Seattle, WA) were also measured using a commercial assay kit.
17. 통계적 분석17. Statistical analysis
각 실험은 적어도 3회씩 독립적으로 반복수행되었다. 값(Values)들은 평균±평균의 표준오차(mean±standard error of the mean(SEM))로 나타내었다. 테스트 그룹간 통계적 유의성의 차이는 one-way analysis of variance (ANOVA) 및 Tukey’post-hoc test에 의하여 평가되었다. 전체 생존률(overall survival rate)의 평가를 위하여, Kaplan-Meier survival analysis가 수행되었다. SPSS for Windows, version 16.0(SPSS, Chicago, IL)을 이용하여 통계적 분석이 수행되었으며, 0.05 미만의 P-값(p <0.05)은 통계적으로 유의미한 것으로 생각되었다. Each experiment was repeated at least three times independently. Values are expressed as means ± standard error of the mean (SEM). Differences in statistical significance between the test groups were assessed by one-way analysis of variance (ANOVA) and Tukey'post-hoc test. To assess the overall survival rate, Kaplan-Meier survival analysis was performed. Statistical analysis was performed using SPSS for Windows, version 16.0 (SPSS, Chicago, IL), and a P-value of less than 0.05 (p <0.05) was considered statistically significant.
<실시예 1>≪ Example 1 >
LPS- 또는 CLP-매개(mediated) HMGB1 분비에 있어서 진저론의 영향Effect of gingerol on LPS- or CLP-mediated HMGB1 secretion
HUVEC 세포의 LPS에 의한 HMGB1의 분비에 대한 진저론의 영향을 확인한 결과, 진저론을 투여하면 용량 의존적으로 HMBG1의 분비를 억제하였으며 10μM에서 최소 유효 농도를 가졌다.(그림 1A) The effect of ginger on the secretion of HMGB1 by LPS in HUVEC cells was inhibited by dose-dependent inhibition of secretion of HMBG1 by gingerol and the lowest effective concentration at 10 μM (Figure 1A).
이러한 활성을 생체 내에서 확인하기 위해 CLP- 유발 패혈증이 일어난 마우스 모델을 사용하여 진저론의 HMGB1 방출을 저해하는 능력을 평가하였고, 진저론으로 처리 한 결과 HMGB1 분비가 유의하게 감소하는 것을 확인하였다. (그림 1B).To confirm these activities in vivo, we evaluated the ability of gingerol to inhibit HMGB1 release using a mouse model in which CLP-induced sepsis occurred, and confirmed that HMGB1 secretion was significantly reduced by treatment with gingerolone. (Figure 1B).
생쥐의 평균 순환 혈액량은 72mL/kg, 사용 된 마우스의 평균 몸무게가 27g, 평균 혈액량이 2mL인 것을 고려하여, 말초 혈액에서 25 또는 50μM 농도가 되도록 상응하는 양의 진저론(0.36 또는 0.72mg/kg)이 투여되었다. Considering that the average circulating blood volume of the mice was 72 mL / kg, the average weight of the mice used was 27 g, and the mean blood volume was 2 mL, the corresponding amount of gingerol (0.36 or 0.72 mg / kg ).
진저론에 의해 HMGB1의 분비가 억제되는 기작을 확인하기 위해, HMGB1의 탈아세틸화 및 SIRT1의 발현을 유도하여 진저론의 영향을 조사하였다. 도 1C에 도시 된 바와 같이, LPS에 의한 자극은 HMGB1의 아세틸화를 증가시켰는데, 이는 주로 진저론의 첨가에 의해 감소되었다. To investigate the mechanism by which gumoderma inhibited the secretion of HMGB1, the effects of gingerol were investigated by inducing deacetylation of HMGB1 and expression of SIRT1. As shown in FIG. 1C, stimulation with LPS increased acetylation of HMGB1, which was mainly reduced by the addition of gingerolone.
SIRT1 발현의 유도에 대한 진저론의 영향을 확인하기위하여 웨스턴 블랏 분석을 수행한 결과, 도 1D에 도시 된 바와 같이, SIRT1 단백질은 4시간 항온처리로 발현됐고, 6시간 후 최대, 8시간까지 유지, 12시간 후에 사라졌다. SIRT1 활성으로 인해 LPS- 활성화 된 HUVEC에서 HMGB1이 탈아세틸화되고 HMGB1 방출이 억제되는지 확인하기 위해 SIRT1 억제제인 sirtinol의 효과를 조사하였다. Western blot analysis was performed to confirm the effect of ginger on the induction of SIRT1 expression. As shown in FIG. 1D, the SIRT1 protein was expressed by incubation for 4 hours and maintained for up to 8 hours after 6 hours , Disappeared after 12 hours. The effect of sirtinol, a SIRT1 inhibitor, was investigated to determine if HMGB1 was deacetylated and HMGB1 release was suppressed in LPS-activated HUVECs by SIRT1 activity.
그림 1C에서 보는 바와 같이, sirtinol의 처리로 진저론의 효과와 반대로, HMGB1의 아세틸화 및 분비가 일어나는 것을 확인하였다. 이 결과는 다음에 기초하여 진저론이 HMGB1 방출을 현저하게 감소시킨다는 것을 시사한다; 1) 진저론은 SIRT1 단백질의 발현을 유의하게 증가시킨다; 2) 진저론에 의해 HMGB1가 탈아세틸화되면 HMGB1의 분비가 유의하게 감소한다; 3) 진저론에 의한 HMGB1 방출 및 아세틸화의 억제는 SIRT1 억제제인 sirtinol에 의해 유의적으로 길항되었다.As shown in Fig. 1C, the sirtinol treatment confirmed the acetylation and secretion of HMGB1, as opposed to the effect of gingerolone. This result suggests that gingerulosis significantly reduces HMGB1 release based on: 1) gingerrone significantly increases the expression of SIRT1 protein; 2) When HMGB1 is deacetylated by gingerolone, secretion of HMGB1 is significantly reduced; 3) The suppression of HMGB1 release and acetylation by gingerolone was significantly antagonized by the SIRT1 inhibitor sirtinol.
다음으로 HUVEC에서 TLR2, TLR4 및 RAGE와 같은 HMGB1 수용체의 발현에 대한 진저론의 영향을 조사하였다. 도 1E에서 보는 바와 같이, HMGB1이 각 수용체를 증가시키고 진저론이 각 수용체의 발현을 감소 시킨다는 것을 확인할 수 있다. 48시간 후 HUVEC에서 진저론의 독성을 조사하기 위해 세포 생존 에세이(Cell viability assay)가 수행되었다. 도 1F에서 보는 바와 같이, 진저론은 100μM의 농도까지, 세포 생존성에 영향을 미치지 않았다. 그러므로 진저론에 의해 HMGB1의 분비가 억제되어 심각한 패혈증 및 패혈성 쇼크 진행 예방에 사용될 수 있음을 확인하였다.Next, we examined the effect of ginger on the expression of HMGB1 receptors such as TLR2, TLR4 and RAGE in HUVEC. As shown in FIG. 1E, it can be seen that HMGB1 increases each receptor and gingerol reduces the expression of each receptor. Cell viability assays were performed 48 hours later to investigate the toxicity of gingerol in HUVEC. As shown in FIG. 1F, gingerol did not affect cell viability up to a concentration of 100 μM. Therefore, it was confirmed that the secretion of HMGB1 is suppressed by gingerolone, and thus it can be used for preventing severe sepsis and septic shock progression.
<실시예 2>≪ Example 2 >
HMGB1-매개 혈관 장벽(vascular barrier) 붕괴에 있어서, 진저론의 효과In HMGB1-mediated vascular barrier collapse, the effect of gingerolone
HMGB1과 LPS는 혈관 장벽 온전성(barrier integrity, 장벽 통합성)을 파괴하는 것으로 잘 알려져 있기 때문에 HUVEC의 장벽 온전성 유지에 대한 진저론의 영향을 평가하기 위해 혈관 투과 에세이(permeability assay)를 수행하였다. Since HMGB1 and LPS are well known to destroy vascular barrier integrity (barrier integrity), a permeability assay was performed to assess the effect of ginger on the barrier integrity of HUVECs .
HUVEC 세포들은 LPS(도 2A, 100ng/mL, 4h) 또는 HMGB1 (도 2B, 1μg/mL, 16h)의 첨가 후에, 진저론으로 6시간동안 처리되었다. 진저론의 처리는 LPS- 또는 HMGB1-에 의해 매개된 장벽 온전성(barrier integrity)의 붕괴를 농도 의존적으로 억제하였다.(도 2A 및 도2B 참조)HUVEC cells were treated with gingerolin for 6 hours after addition of LPS (Fig. 2A, 100 ng / mL, 4 h) or HMGB1 (Fig. 2B, 1 ug / mL, 16 h). Treatment of gingerol suppressed concentration-dependent disruption of barrier integrity mediated by LPS- or HMGB1- (see Figures 2A and 2B).
상기와 같은 혈관장벽 보호효과를 in vivo상에서 확인하기 위하여, 마우스에서 HMGB1에 의해 매개된 혈관 투과성(vascular permeability)을 평가하였다. 도 2C에서 보는 바와 같이, 진저론의 처리는 HMGB1에 의해 유도된 복막 염료 누설(peritoneal leakage)을 현저하게 억제하는 결과를 나타낸다.In order to confirm the vascular barrier protection effect as described above in vivo , HMGB1-mediated vascular permeability was evaluated in mice. As shown in FIG. 2C, treatment with gingerolone resulted in marked inhibition of peritoneal leakage induced by HMGB1.
다음으로 HMGB1 수용체의 일반적인 신호 전달 타겟인 p38의 활성화가 진저론의 영향을 받는지 여부를 조사하였다. 이를 위해 HMGB1을 활성화한 뒤, HUVEC에 진저론을 투여하였다. 도 2D에서 보는 바와 같이, HMGB1은 인산화 된 p38의 발현을 증가 시켰으며, 이는 진저론 처리에 의해 명백하게 감소되었다. 또한, 진저론 (20 또는 30μM) 처리는 조밀한 F- actin 고리를 형성하면서 HMGB1 유도된 세포 간극의 형성을 억제하였다 (도 2E). 진저론에 의한 HMGB1-유도 투과성 및 p38 활성화가 감소된 결과를 통해 진저론이 항패혈증 약물로서의 유망함을 확인하였다.Next, we investigated whether the activation of p38, a common signaling target of the HMGB1 receptor, was affected by gingerolone. For this, HMGB1 was activated and then gingerol was administered to HUVEC. As shown in Figure 2D, HMGB1 increased the expression of phosphorylated p38, which was apparently reduced by gingerol treatment. In addition, gingerol (20 or 30 μM) treatment inhibited the formation of HMGB1-induced cell gaps while forming dense F-actin rings (FIG. 2E). As a result of reduced HMGB1-induced permeability and p38 activation by gingerolone, gingerolone was promising as an antiepileptic drug.
<실시예 3>≪ Example 3 >
HMGB1-매개 CAM(cell-adhesion molecule)들의 발현, 인간 호중구 부착 및 이동에 대한, 진저론의 효과The effects of gingerolone on expression of HMGB1-mediated CAM (cell-adhesion molecules), adhesion and migration of human neutrophils
이전의 연구들은 HMGB1이 내피세포의 표면에 ICAM-1, VCAM-1 및 E-selectin과 같은 CAM들의 발현을 증가시킴으로서 염증성 반응을 매개하고, 그 때문에 내피(endothelium)를 가로질러 염증 부위에 백혈구의 이동 및 부착을 촉진한다고 보고하였다. 본 연구에 따르면, HMGB1은 VCAM-1, ICAM-1 및 E-selectin의 표면 발현 증가를 유도하며(도 3A 참조), 진저론은 상기 CAM들의 발현을 억제하는데, 이는 CAM들의 발현에 대한 진저론의 억제효과가 상기 화합물들에 의해 HMGB1 signaling pathway의 약화를 통하여 매개된다는 것을 제시한다.Previous studies have shown that HMGB1 mediates the inflammatory response by increasing the expression of CAMs such as ICAM-1, VCAM-1 and E-selectin on the surface of endothelial cells, thereby causing inflammation of the white blood cells across the endothelium As well as to promote migration and adhesion. HMGB1 induces increased surface expression of VCAM-1, ICAM-1 and E-selectin (see FIG. 3A), and gingerol suppresses the expression of the CAMs, ≪ / RTI > is mediated through the attenuation of the HMGB1 signaling pathway by these compounds.
게다가, 증가된 CAM들의 발현은 HMGB1에 의해 활성화된 내피세포에 대한 인간 호중구의 증가된 결합 및 이에 이어서 발생되는 호중구 이동(migration)과 부합된다. 진저론의 처리는 인간 호중구의 부착 및 차후 활성화된 내피세포를 가로지르는 이동(migration)을 농도 의존적으로 감소시키는 결과를 보였다(도 3B, 도 3C, 도 3E 참조). In addition, the increased expression of CAMs is consistent with increased binding of human neutrophils to HMGB1-activated endothelial cells and subsequent neutrophil migration. Treatment of gingerol resulted in the concentration-dependent reduction of attachment of human neutrophils and migration across later activated endothelial cells (see Figures 3B, 3C and 3E).
이러한 결과는 진저론이 단지 내피세포에서 내독소-매개(endotoxin-mediated) HMGB1의 분비를 억제하는 것 뿐만 아니라, HMGB1의 분비에 의해 야기된 전염증성 시그날링 효과(proinflammatory signaling effect)를 억제하며, 그 때문에 핵 사이토카인(nuclear cytokine)들에 의한 염증성 경로(inflammatory pathway)의 증폭과정을 억제한다는 것을 제시한다.These results suggest that gingerolone not only inhibits the secretion of endotoxin-mediated HMGB1 in endothelial cells but also suppresses the proinflammatory signaling effect caused by HMGB1 secretion, Thus inhibiting the amplification of the inflammatory pathway by nuclear cytokines.
이러한 효과를 in vivo 상에서 확인하기 위하여, 마우스에서 HMGB1-에 의해 유도된 백혈구 이동을 조사하였다. HMGB1은 마우스의 복막강(peritoneal cavity)으로 백혈구의 이동을 촉진하였고, 진저론의 처리는 이러한 이동을 현저히 억제하였다(도 3D 참조). To confirm this effect in vivo , leukocyte migration induced by HMGB1- was investigated in mice. HMGB1 promoted leukocyte migration into the peritoneal cavity of mice, and treatment with gingerol significantly inhibited this migration (see Figure 3D).
백혈구 및 내피세포 사이의 상호작용 조절에 대한 연구를 위해, in vitro상에서 CAM들에 대한 실험이 널리 수행된다. 본 연구에서 진저론은 HMGB1에의해 유도되는 VCAM-1, ICAM-1, 및 E-selectin 수준(level)을 감소하는 결과를 나타냈으며, 이는 활성화된 내피에 백혈구가 부착 및 이동하는 것을 진저론이 억제 한다는 것을 제시한다. In order to study the regulation of interactions between leukocytes and endothelial cells, experiments on CAMs are performed in vitro. In this study, gingerol reduced VCAM-1, ICAM-1, and E-selectin levels induced by HMGB1, indicating that leukocyte adhesion and migration to activated endothelium was due to ginger .
<실시예 4><Example 4>
HMGB1에 의해 자극된 TNF-α/IL-6의 생성 및 NF-κB/ERK의 활성화에 대한, 진저론의 효과Effect of Gingerol on HMGB1-stimulated TNF-α / IL-6 production and activation of NF-κB / ERK
전염증성 사이토카인들(TNF-α, IL-1α 및 IL-1β, IL-12, 및 IL-6)은 감염에 대항하여 효과적인 염증 프로세스를 촉발하는데 있어 필요하지만, 이들의 과잉 생성은 여러 장기의 기능부전(multiple organsystem dysfunction) 및 사망률과 관계되어있다. 그러므로 우리는 HMGB1에 의해 매개된 전염증성 사이토카인의 생성에 대한 진저론의 영향을 조사하였다. 본 실험결과에 따르면, HMGB1으로 자극된 내피세포에서 IL-1α및 IL-1β(데이터 미도시), TNF-α(도 4A 참조), 및 IL-6(도 4B참조)의 수준이 증가되었으나, 이러한 증가 현상은 진저론의 처리에 의하여 현저히 감소되었으며(도 4A 및 도4B 참조), 이는 진저론이 인간 내피세포에서 전염증성 반응의 유도와 관계된 가장 중요한 신호(시그널, signal)를 조절한다는 사실을 나타낸다. Although proinflammatory cytokines (TNF-a, IL-l [alpha] and IL-l [beta], IL-12 and IL-6) are required to trigger an effective inflammatory process against infection, Multiple organsystem dysfunction and mortality. Therefore, we investigated the effect of ginger on the production of HMGB1-mediated proinflammatory cytokines. The levels of IL-1α and IL-1β (data not shown), TNF-α (see FIG. 4A) and IL-6 (see FIG. 4B) were increased in endothelial cells stimulated by HMGB1, This increase was markedly reduced by the treatment of gingerol (see FIGS. 4A and 4B), indicating that gingerolone regulates the most important signals (signals) involved in the induction of proinflammatory responses in human endothelial cells .
게다가, NF-κB 및 ERK1/2의 활성화는 전염증성 반응의 촉발을 위해 필요하다. HMGB1은 혈관 염증성 반응에 있어서 NF-κB 및 ERK 1/2를 활성화하는 것으로 알려져있다. 그러므로 우리는 HMGB1에 의한 NF-κB 및 ERK 1/2의 활성화에 대하여 진저론의 영향을 평가하였다. 도 4C 내지 도 4E에서 보는 바와 같이, HMGB1의 처리는 NF-κB 및 ERK 1/2의 활성화를 증가시켰고, 이러한 증가는 진저론의 처리에 의하여 현저하게 감소괴는 결과를 나타냈다. 염증성 반응은 혈관 손상의 발병에 있어서 중요한 요소이고, 내피 기능이상(endothelial dysfunction)은 혈관의 염증성 병변 생성 동안에 백혈구의 소집(recruitment)과 특히 관련되어있다. TNF-α(Tumor necrosis factor-α) 및 NF-κB(nuclear factor-κB)는 혈관 염증성 프로세스에 관련된 중요한 매개자(mediator)들이다. NF-κB는 잘 알려진 전-염증성 전사 인자(transcriptional factor)이다. NF-κB의 활성화는 TNF-α 및 IL-1β와 같은 전-염증성 사이토카인들에 대한 반응으로서 발생된다. NF-κB 시그날링 경로의 억제가 상당한 혈관 보호 효과에 기여하며, 이는 질환 동물 모델에서 혈관 질환이 진행되는 것을 막거나 지연시킨다는 것을 제시하는 상당한 증거들이 존재한다. 그러므로, 혈관 내피세포에서 TNF-α의 생성 및 NF-κB 활성화를 막는 것은 혈관염증질환의 치료를 위한 유망한 치료적 타겟으로 고려되고 있으며, 진저론은 염증 매개자 생산 및 NF-κB 경로를 억제하는 것을 통하여 항염증 효과를 나타낸다.In addition, activation of NF-kB and ERK1 / 2 is necessary for triggering proinflammatory responses. HMGB1 is known to activate NF-κB and
<실시예5>≪ Example 5 >
CLP-유도 패혈증 사망률에 있어서, 진저론의 보호 효과In CLP-induced sepsis mortality, the protective effect of gingerolone
패혈증은 심각한 감염에 대한 전신성 반응(systemic response)으로서, 장기 기능부전(organ dysfunction), 저혈류(hypoperfusion) 또는 저혈압(hypotension)과 관련되어있을 때 나쁜 예후를 보인다. 전술한 실험결과들에 기초하여, CLP-유도 패혈증 마우스 모델에서 진저론의 처리가 사망률(mortality)을 감소시킬 수 있을 것이라는 가정을 하였다. 진저론이 CLP-유도 패혈증 치사(lethality)로부터 마우스를 보호할 수 있을지 조사하기 위하여, 20 마리의 마우스들을 대상으로 CLP 수술 후, 진저론을 주입하였다. CLP 수술 24시간 후에, 실험동물들은 떨림(shivering), 거친 털(bristled hair) 및 쇄약(weakness)과 같은 패혈증 증상을 나타냈다. CLP 후 12시간째에 진저론(0.36 또는 0.72mg/kg)를 1회 투여하는 것은 CLP에 의한 죽음을 막지 못하였다(데이터 미도시). 그러므로, 2회 걸쳐 상기 화합물들을 투여하였으며(1차 투여는 CLP 후 12시간째에, 2차 투여는 CLP 후 50시간째에), Kaplan-Meier 생존 분석 결과 상기 2회 처리로 인하여 30 내지 60%로 생존률이 증가되었다(p < 0.05 versus CLP, 도 5A참조). 이러한 결과는, HMGB1의 분비와 HMGB1-매개 염증 반응의 저해는 패혈증 및 패혈성 쇼크의 관리를 위한 치료적 전략임을 제시한다. Sepsis is a systemic response to a serious infection that has a bad prognosis when it is associated with organ dysfunction, hypoperfusion, or hypotension. Based on the above-described experimental results, it was assumed that treatment of gingerolone in a CLP-induced septic mouse model would reduce mortality. To investigate whether ginger rinses could protect mice from CLP-induced sepsis lethality, ginger rats were injected after CLP surgery on 20 mice. After 24 hours of CLP surgery, the experimental animals exhibited symptoms of sepsis such as shivering, bristled hair, and weakness. A single dose of gingerol (0.36 or 0.72 mg / kg) at 12 hours after CLP did not prevent death by CLP (data not shown). Therefore, the compounds were administered twice (the first dose at 12 hours after CLP, the second dose at 50 hours after CLP) and the Kaplan-Meier survival analysis showed that 30-60% (P < 0.05 versus CLP, see Fig. 5A). These results suggest that inhibition of HMGB1 secretion and HMGB1-mediated inflammatory response is a therapeutic strategy for the management of sepsis and septic shock.
<실시예 6>≪ Example 6 >
CLP-유도 조직 손상에 대한, 진저론의 보호 효과Protective Effect of Gingerol on CLP-Induced Tissue Injury
CLP-유도성 죽음에 대한 진저론의 보호효과를 확인하기 위하여, CLP-유도 폐 손상에 대한 진저론의 효과를 조사하였다. CLP 수술은 간질과 폐포 공간에 염증 세포가 다량으로 침투하여 간질 부종을 가져 왔고 폐 구조를 심하게 손상시켰다. 이러한 부종 및 손상은 진저론으로 처리 한 CLP 마우스에서 감소했다 (도 6A 및 도6B 참조). In order to confirm the protective effect of gingerolone on CLP-induced death, the effect of gingerolone on CLP-induced lung injury was investigated. CLP surgery resulted in epileptic seizures and massive infiltration of inflammatory cells into the epilepsy and alveolar space, severely impairing lung architecture. These edema and damage were reduced in CLP mice treated with gingerolone (see Figures 6A and 6B).
패혈증에 의한 전신 염증은 종종 패혈증의 주요 표적 기관인 간 및 신장에서 다발성 장기 부전을 일으킨다. CLP는 혈장에서 간 손상의 마커인 ALT, AST (도 6C 참조), 신장 손상의 마커인 크레아티닌 및 BUN (도 6D, 도 6E 참조)을 현저히 증가시켰으나, 이러한 마커들은 진저론에 의해 감소되었다. 그리고 CLP수술 된 마우스에서 조직 손상시 혈액 중으로 흘러나와 상승한 LDH 또한, 진저론에 의해 감소되었다.(그림 6F)Systemic inflammation due to sepsis often causes multiple organ failure in the liver and kidneys, the main target organ of sepsis. CLP markedly increased ALT, AST (see FIG. 6C), which is a marker of liver damage in plasma, creatinine, and BUN (see FIG. 6D, FIG. 6E), which are markers of kidney damage, but these markers were reduced by gingerolone. In addition, elevated LDH released into the bloodstream during tissue injury in CLP-treated mice was also reduced by gingerol (Figure 6F).
한편, 내분비계 장애는 패혈증이 심한 경우 흔하게 발생하며 사망 위험이 증가한다. 갑상선 호르몬의 이상 또한 패혈증 환자에서 매우 흔하다. T3, T4, TSH를 포함한 기준보다 낮은 갑상선 호르몬 수치는 유사한 중증도의 패혈증이 없는 환자와 비교하여 패혈증 환자에서 유의하게 낮을 수 있으며, 이러한 이상은 패혈증 환자의 중증과 관련이 있다. CLP로 유발된 패혈증 상태에서 T3, T4 및 TSH 수준에 대한 진저론의 영향을 조사한 결과, CLP-유도 패혈증 마우스와 비교한 결과 진저론 처리로 회복된 마우스에서 T3, T4 및 TSH의 수준이 더 낮았다. (도 6G 내지 도 6I). 이러한 결과는, 진저론의 투여가 패혈증으로 유도된 내분비계 장애 치료를 위한 효과적인 치료전략임을 제시한다.On the other hand, endocrine disorders are common when severe sepsis occurs and the risk of death increases. Thyroid hormone abnormalities are also very common in sepsis patients. Thyroid hormone levels lower than the standard including T3, T4, and TSH may be significantly lower in patients with sepsis compared to patients without similar severity of sepsis, and these abnormalities are associated with severity in sepsis patients. Studies of the effect of ginger on T3, T4 and TSH levels in the CLP-induced sepsis state revealed that the levels of T3, T4 and TSH were lower in mice restored by gingerol treatment compared with CLP-induced septic mice . (Figs. 6G to 61). These results suggest that administration of gingerolone is an effective treatment strategy for the treatment of endocrine system disorders induced by sepsis.
이상 살펴본 바와 같이, 본 발명은 진저론을 유효성분으로 포함하는 패혈증의 예방 또는 치료용 조성물에 관한 것으로, 보다 상세하게는 진저론을 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 예방, 개선 또는 치료용 (약학적 또는 식품)조성물에 관한 것이다. As described above, the present invention relates to a composition for preventing or treating septicemia comprising gingerol as an active ingredient, and more particularly to a composition for preventing or treating septicemia or septic shock comprising gingerol as an active ingredient (Pharmaceutical or food) composition.
본 발명의 진저론은 패혈증의 주요 매개인자인 HMGB1(High mobility group box 1)의 분비를 억제하고 상기 HMGB1과 관련된 전염증성 시그날링(proinflammatory signaling)을 억제하는 효과를 지니며, 실제로 in vivo 패혈증 동물 모델에서 패혈증의 치료효과가 현저하므로 산업상 이용가능성이 높다. The ginger polysaccharide of the present invention suppresses secretion of HMGB1 (high mobility group box 1), which is a major mediator of sepsis, and inhibits proinflammatory signaling associated with HMGB1. In fact, in vivo sepsis Since the treatment effect of sepsis is remarkable in the model, the possibility of industrial use is high.
Claims (3)
<화학식 1>
A pharmaceutical composition for preventing or treating septicemia or septic shock mediated by HMGB1 (High mobility group box 1) containing gingerol as an active ingredient represented by formula (1).
≪ Formula 1 >
<화학식 1>
A food composition for preventing or ameliorating septicemia or septic shock mediated by HMGB1 (High mobility group box 1) containing gingerol as an active ingredient represented by formula (1).
≪ Formula 1 >
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KR1020170048759A KR101909954B1 (en) | 2017-04-14 | 2017-04-14 | Composition for prevention or treatment of sepsis or septic shock comprising Zingerone |
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KR1020170048759A KR101909954B1 (en) | 2017-04-14 | 2017-04-14 | Composition for prevention or treatment of sepsis or septic shock comprising Zingerone |
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KR101909954B1 true KR101909954B1 (en) | 2018-10-19 |
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KR1020170048759A KR101909954B1 (en) | 2017-04-14 | 2017-04-14 | Composition for prevention or treatment of sepsis or septic shock comprising Zingerone |
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KR (1) | KR101909954B1 (en) |
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2017
- 2017-04-14 KR KR1020170048759A patent/KR101909954B1/en active IP Right Grant
Non-Patent Citations (1)
Title |
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Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway, European Journal of Pharmacology, 772, 108-114(2016.)* |
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