KR20200120057A - Pharmaceutical composition for treating ototoxicity hearing loss by additional effect of cisplatin comprising alpha-lipoic acid - Google Patents
Pharmaceutical composition for treating ototoxicity hearing loss by additional effect of cisplatin comprising alpha-lipoic acid Download PDFInfo
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- KR20200120057A KR20200120057A KR1020190042439A KR20190042439A KR20200120057A KR 20200120057 A KR20200120057 A KR 20200120057A KR 1020190042439 A KR1020190042439 A KR 1020190042439A KR 20190042439 A KR20190042439 A KR 20190042439A KR 20200120057 A KR20200120057 A KR 20200120057A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
- A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
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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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
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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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
-
- 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
- A23V2250/00—Food ingredients
- A23V2250/02—Acid
- A23V2250/026—Alpha lipoic acid, thioctic acid
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Mycology (AREA)
- Nutrition Science (AREA)
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Description
본 발명은 알파-리포익산(alpha-lipoic acid)을 유효성분으로 함유하는 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물 및 건강기능식품에 관한 것이다.The present invention relates to a pharmaceutical composition and a health functional food for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid as an active ingredient.
1960 년대에 항암 효과가 발견된 이후 시스플라틴(cis-diamminedichloroplatinum II)은 난소, 폐, 방광 등에서 발견되는 고형 종양에 유효한 화학 요법 약물로 널리 사용되었다.Since its anticancer effect was discovered in the 1960s, cis-diamminedichloroplatinum II has been widely used as an effective chemotherapy drug for solid tumors found in the ovaries, lungs, and bladder.
시스플라틴(cisplatin)은 확산(diffusion) 또는 운반체(transporter)에 의해 세포로 들어가고 수화(hydrate)되어 세포 내부의 염화 농도(chloride ions)를 낮추기 때문에 염화 이온(chloride ions)을 방출하게 한다. 수화된 형태의 시스플라틴은 핵과 미토콘드리아 DNA의 뉴클레오타이드와 교차결합(cross-links)하여 부가물(adducts)을 형성하여 암세포의 무제한적인 복제를 억제시킨다. Cisplatin enters the cell by diffusion or transporter and is hydrated to lower the chloride concentration inside the cell, thereby releasing chloride ions. The hydrated form of cisplatin cross-links with the nucleotides of the nucleus and mitochondrial DNA to form adducts, thereby inhibiting the unlimited replication of cancer cells.
그러나, 시스플라틴을 이용한 임상 치료는 정상 조직의 부작용으로서 신독성(nephrotoxicity), 신경독성(neurotoxicity) 및 이독성(ototoxicity)을 유발하기 때문에 사용에 제한이 있다. 특히 시스플라틴에 의해 유발된 이독성(ototoxicity)은 돌이킬 수 없는 감각신경성 청력손실, 즉 난청(hearing loss)을 유발시킬 수 있으며, 특히 소아 집단에서 더욱 심각한 문제가 된다. However, clinical treatment using cisplatin is limited in use because it causes nephrotoxicity, neurotoxicity, and ototoxicity as side effects of normal tissues. In particular, cisplatin-induced ototoxicity can cause irreversible sensorineural hearing loss, that is, hearing loss, and is a more serious problem, especially in the pediatric population.
난청(hearing loss)은 환자의 삶의 질을 크게 저해시킬 수 있다. 특히 언어 습득 및 소아 환자의 말하기와 같은 사회 발전에 상당한 영향을 줄 수 있다.Hearing loss can significantly impair a patient's quality of life. In particular, it can have a significant impact on social development, such as language acquisition and speaking of pediatric patients.
비록 시스플라틴의 이독성을 일으키는 근원적인 분자 기전이 완전하게 밝혀지지 않았지만, 이전 연구에서는 시스플라틴이 정상 세포에서 사멸 수용체 경로(death receptor pathway), 소포체 스트레스 경로(endoplasmic reticulum-stress pathway) 및 미토콘드리아 반응성 산소종 (ROS) 생성 경로(mitochondrial reactive oxygen species (ROS)-generating pathway)를 활성화시킴을 보여 주었고, 결국 세포 죽음으로 이어짐을 밝힌 바 있다.Although the underlying molecular mechanisms responsible for cisplatin ototoxicity have not been fully elucidated, previous studies have shown that cisplatin is the death receptor pathway, endoplasmic reticulum-stress pathway, and mitochondrial reactive oxygen species in normal cells. It has been shown to activate the (ROS) production pathway (mitochondrial reactive oxygen species (ROS)-generating pathway), which in turn leads to cell death.
과도한 ROS의 생성은 시스플라틴으로부터 유도된 이독성 난청의 주요 원인으로 여겨지며, 또한 이는 DNA에 대한 시스플라틴의 직접적인 공격으로 간주된다.Excessive ROS production is believed to be a major cause of cisplatin-induced ototoxic hearing loss, and it is also considered a direct attack of cisplatin on DNA.
특히 달팽이관(cochlea) 내의 코르티(corti) 기관, 나선형 신경절(spiral ganglion) 및 측벽(lateral wall)은 강력한 ROS 생성을 위한 시스플라틴의 주요 표적이라고 제안되어 왔다.In particular, the corti organs, spiral ganglions and lateral walls in the cochlea have been proposed as major targets of cisplatin for the production of strong ROS.
알파-리포익산(alpha-lipoic acid; ALA)은 티옥산(Thioctic acid) 또는 1,2-디치올란-3-펜타노익산(1,2-dithiolane-3-pentanoic acid; C8H14O2S2)으로 널리 알려져 있으며, 이는 세포막, 세포질 및 세포 외 공간에 광범위하게 분포하고, 물과 지질에서 용해되며, 미토콘드리아 탈수소 효소 반응에서 필수적인 보조 인자이다. Alpha-lipoic acid (ALA) is Thioctic acid or 1,2-dithiolane-3-pentanoic acid; C 8 H 14 O 2 S 2 ), which is widely distributed in the cell membrane, cytoplasm and extracellular space, is soluble in water and lipids, and is an essential cofactor in mitochondrial dehydrogenase reactions.
알파-리포익산(alpha-lipoic acid; ALA)의 보호 작용(protective action)은 1959년 Rosenberg와 Culik에 의해 처음 보고되었으며, Rosenberg와 Culik은 비타민 C와 비타민 E 결핍으로 인한 괴혈병의 증상을 줄이는 역할이 발견하였다.The protective action of alpha-lipoic acid (ALA) was first reported by Rosenberg and Culik in 1959, and Rosenberg and Culik have a role in reducing the symptoms of scurvy caused by vitamin C and vitamin E deficiency. I found it.
나중에 알파-리포익산(ALA)이 비타민 C와 비타민 E 및 세포내 환원된 글루타티온(GSH)을 포함해 체내의 내인성 항산화 물질(endogenous antioxidants)을 재생하는 능력이 있다는 것이 밝혀졌다.It was later discovered that alpha-lipoic acid (ALA) has the ability to regenerate endogenous antioxidants in the body, including vitamin C and vitamin E and intracellular reduced glutathione (GSH).
최근에는 알파-리포익산(ALA)이 레톡스 커플(redox couple)로서 환원된 형태의 디하이드로 리포익산(dihydrolipoic acid)과 함께 보편적인 항산화제로서 상당한 주목을 받고 있다.Recently, alpha-lipoic acid (ALA) is receiving considerable attention as a universal antioxidant along with dihydrolipoic acid in a reduced form as a redox couple.
한편 시스플라틴 화학 요법이 암 환자에게 필수적이지만, 종래의 연구는 알파-리포익산(ALA)의 예방적 차원에서의 전처리 효과(protective pre-treatment effects)에만 초점이 맞추어져 있었다.Meanwhile, cisplatin chemotherapy is essential for cancer patients, but previous studies have focused only on the protective pre-treatment effects of alpha-lipoic acid (ALA).
또한 종래의 연구는 신독성(nephrotoxicity) 및 신경독성(neurotoxicity)을 포함한 시스플라틴에 의해 유발된 주요 부작용이 동물 모델에서의 알파-리포익산(ALA) 사전 투여에 의해 효과적으로 예방된다는 것을 입증하였을 뿐이며, 현재까지는 시스플라틴에 의해 유발된 이독성(ototoxicity) 난청(hearing loss)을 근본적으로 치료하는 치료제에 대해서는 전혀 보고되어 있지 않다.In addition, conventional studies have only demonstrated that major side effects caused by cisplatin, including nephrotoxicity and neurotoxicity, are effectively prevented by pre-administration of alpha-lipoic acid (ALA) in animal models. Until now, no treatment has been reported for the treatment of ototoxicity and hearing loss caused by cisplatin.
본 발명의 발명자들은 시스플라틴에 의한 청력 손실이 된 마우스 모델에서 알파-리포익산(ALA)이 미치는 영향을 조사하였고, in vitro에서 청각유모세포를 이용하여 알파-리포익산(ALA)에 의해 프로아폽토틱 단백질(proapoptotic protein) Bax 유전자 또는 단백질 발현은 감소하고, 안티아폽토틱 단백질(antiapoptotic protein) Bcl-2 유전자 또는 단백질 발현은 증가함으로써 미토콘드리아의 기능 장애를 개선시켜 이독성에 의한 난청을 줄여주는 것을 확인하였다. 또한 항산화 효소인 글루타치온의 산화-환원 반응에 직접 관여하여 활성산소(ROS)를 낮춘다는 것을 확인하였다. 기존에 밝혀진 알파-리포익산(ALA)에 의한 예방효과 뿐 아니라 후처리를 통한 치료효과를 확인할 수 있었으며, 이러한 알파-리포익산(ALA)의 후처리 효과는 시스플라틴에 의해 유발된 난청을 치료하는 치료제 개발로의 가능성을 의미한다. 이를 통해 본 발명자들은 알파-리포익산(ALA)에 의해 활성화된 항세포사멸(antiapoptotic) 경로를 밝히면서 본 발명을 완성하기에 이르렀다.The inventors of the present invention investigated the effect of alpha-lipoic acid (ALA) in a mouse model of hearing loss caused by cisplatin, and proapoptotic by alpha-lipoic acid (ALA) using auditory hair cells in vitro . It was confirmed that the expression of the protein (proapoptotic protein) Bax gene or protein decreased, and the expression of the antiapoptotic protein Bcl-2 gene or protein increased, thereby improving mitochondrial dysfunction and reducing hearing loss caused by ototoxicity. . In addition, it was confirmed that the antioxidant enzyme glutathione is directly involved in the oxidation-reduction reaction to lower free radicals (ROS). As well as the previously discovered effects of alpha-lipoic acid (ALA), as well as the therapeutic effect through post-treatment, the post-treatment effect of alpha-lipoic acid (ALA) is a therapeutic agent that treats hearing loss caused by cisplatin. It means the possibility of development. Through this, the inventors of the present invention have come to complete the present invention by revealing an antiapoptotic pathway activated by alpha-lipoic acid (ALA).
본 발명은 알파-리포익산(ALA)을 유효성분으로 함유하는 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물 또는 건강기능식품을 제공하고자 한다.The present invention is to provide a pharmaceutical composition or health functional food for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.
본 발명은 알파-리포익산(ALA)을 유효성분으로 함유하는 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.
또한, 알파-리포익산(ALA)을 유효성분으로 함유하는 시스플라틴에 의한 이독성 난청 개선용 건강기능식품을 제공한다. In addition, it provides a health functional food for improving ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.
본 발명은 알파-리포익산(ALA)이 시스플라틴에 의한 이독성 난청이 발병한 마우스에서 청력 검사시 청력 손실을 확연히 감소시켰으며, 마우스의 내이를 추출하여 형태학적 관찰시 청각유모세포와 나선형 신경절 세포의 손상을 막아 주었고, 청각유모세포에서 Bax 유전자 또는 단백질 발현은 감소시키고, Bcl-2 유전자 또는 단백질 발현은 증가시킴으로써 미토콘드리아의 기능 장애를 개선시켜 이독성에 의한 난청을 줄여주었으며, 항산화 효소인 글루타치온의 산화-환원 반응에 직접 관여하여 활성산소(ROS)를 낮춘다는 것을 확인함으로써, 본 발명의 알파-리포익산은 시스플라틴 부작용으로 나타나는 이독성 난청의 치료 조성물 또는 개선용 건강기능식품으로 널리 사용될 수 있다. In the present invention, alpha-lipoic acid (ALA) significantly reduced hearing loss during hearing test in mice with ototoxic hearing loss caused by cisplatin, and auditory hair cells and spiral ganglion cells when morphological observation by extracting the inner ear of the mouse In the auditory hair cells, the expression of the Bax gene or protein was reduced, and the expression of the Bcl-2 gene or protein was increased to improve mitochondrial dysfunction, thereby reducing hearing loss due to ototoxicity, and the antioxidant enzyme glutathione. By confirming that it directly participates in the oxidation-reduction reaction and lowers the active oxygen (ROS), the alpha-lipoic acid of the present invention can be widely used as a therapeutic composition for ototoxic hearing loss or a health functional food for improvement that appears as a side effect of cisplatin.
도 1은 시스플라틴 주입 마우스(cisplatin-injected mice)에 ALA 전처리 또는 ALA 후처리하고 ABR 측정하여 얻은 청력 검사 결과이다(*p <0.05 vs 시스플라틴 단독, 대조군; control group, 시스플라틴 군; CP group, ALA 전처리 군; ALA pre-treatment group, ALA 후처리 군; ALA post-treatment group, ALA 군; ALA group, ABR; auditory brainstem response).
도 2는 ALA 전처리 또는 ALA 후처리한 시스플라틴 주입 마우스(cisplatin-injected mice)의 내부 귀(inner ear)의 조직학적 분석 결과로서(n = 3), 도 2A는 대조군 마우스의 내이의 Basal turn 모습을 나타낸 것이고, 도 2B는 혈관선조(stria vascularis)[위] 및 나선형 신경절(spiral ganglion)[아래]을 고배율로 관찰한 것이고(이중 화살표(↔)는 혈관선조의 두께, 별표(*)는 나선형 신경절 세포의 퇴행을 나타낸 것이고, 스케일 바는 50 μm임), 도 2C는 혈관선조 두께(단위: ㎛)의 정량적 비교한 결과이며, 도 2d는 기저부(basal), 중간(middle) 및 정점(apical) turn에서 각 그룹 간의 나선형 신경절 세포수를 상대적으로 비교한 결과이다(*p <0.05 vs 시스플라틴 단독).
도 3은 시스플라틴 주입 마우스(cisplatin-injected mice)에서 시스플라틴에 의해 유도된 청각유모세포 손상에 대한 ALA 투여 전후 치료 효과를 확인한 결과로서, 도 3A는 유모세포의 생존이나 변성을 확인하기 위해 내부 유모세포와 외부 유모세포를 포함한 코르티 기관의 Phalloidin 염색(적색) 결과를 타나낸 것이고(흰색 별표(*)는 퇴행성 유모세포를 나타낸 것이고, 스케일 바는 50 μm임), 도 3B는 각 그룹의 달팽이관(cochlea)의 기저부(basal), 중간(middle) 및 정점(apical) 회전(turn)에서 내부 및 외부 유모세포의 세포수를 히스토그램 정량화한 결과이다(*p <0.05 vs 시스플라틴 단독).
도 4는 시스플라틴 처리 HEI-OC1 세포(cisplatin-treated HEI-OC1 cells)에서 ALA 처리가 세포 생존 능력에 미치는 영향을 확인한 결과로서, 도 4A는 HEI-OC1 세포를 0.5, 1, 2, 3 및 4 mM의 ALA와 30 시간 동안 배양하고 세포 독성을 MTT assay로 측정한 결과이고, 도 4B는 HEI-OC1 세포를 30 분 동안 30 μM 시스플라틴의 처리한 후 1 시간 동안 0.5 내지 4 mM의 ALA로 처리하고 세포 독성을 MTT assay로 측정한 결과이다(*p <0.05 vs 시스플라틴 단독).
도 5는 시스플라틴 처리 HEI-OC1 세포(cisplatin-treated HEI-OC1 cells)에서 ALA가 세포주기 정지(cell cycle arrest) 및 세포사멸(apoptosis)에 미치는 영향을 확인한 결과로서, 도 5A는 유세포분석법(flow cytometry)에 의한 세포주기 분석 결과이고, 도 5B는 CP 단독 처리군과 ALA 단독 처리군 사이의 sub-G0/G1 비율을 비교한 결과이고, 도 5C는 세포사멸 세포(apoptotic cells)를 검출하기 위한 TUNEL assay 분석한 결과이고(형광 현미경(fluorescence microscope) 관찰 시, DNA-녹색, 핵-파란색으로 관찰되고, 스케일 바는 100 μm임), 도 5D는 시스플라틴 및 ALA로 처리한 세포에서 caspase-3 발현을 확인하기 위한 웨스턴 블롯(Western blot) 분석 결과이고, 도 5E는 덴시토미트리(densitometry)를 사용하여 caspase-3:β-actin 비율을 측정한 결과이다(*p <0.05 vs 시스플라틴 단독).
도 6은 시스플라틴 처리된 세포에서 ALA의 ROS 제거능(scavenging)을 나타낸 결과로서, 도 6A는 DCFH-DA probe를 이용한 세포 내 ROS 수치(level)를 측정한 결과이고(형광 강도는 flow cytometer를 사용하여 검출), 도 6B는 상대적인 FACS 형광 강도(FACS fluorescence intensities)를 측정한 결과이다(*p <0.05 vs 시스플라틴 단독).
도 7은 시스플라틴에 의한 Bax 매개 세포사멸(Bax-mediated apoptosis)에 대한 ALA의 항세포사멸 효과(antiapoptotic effect)를 확인한 결과로서, 도 7A는 로딩 대조군(loading control)인 β-actin과 Bax의 웨스턴 블롯 분석 결과이고(n = 3 per lane), 도 7B는 Bax와 β-actin의 상대적 발현 비율을 덴시토미트리(densitometry)를 이용하여 측정한 결과이고, 도 7C는 로딩 대조군(loading control)인 β-actin과 Bcl-2의 웨스턴 블롯 분석 결과이고(n = 3 per lane), 도 7D는 Bcl-2와 β-actin의 상대적 발현 비율을 덴시토미트리(densitometry)를 이용하여 측정한 결과이다(*p <0.05 vs 시스플라틴 단독).
도 8은 ALA 처리 전 또는 처리 후의 세포에서 항산화효소 GPx 및 GR의 발현 수준을 나타낸 결과로서, 도 8A는 로딩 대조군(loading control)인 β-actin와 GPx의 웨스턴 블롯 분석 결과이고(n = 3 per lane), 도 8B는 GR 및 β-actin의 상대적인 발현 비율을 덴시토미트리(densitometry)를 이용하여 측정한 결과이고, 도 8C는 로딩 대조군(loading control)인 β-actin와 GR의 웨스턴 블롯 분석 결과이고(n = 3 per lane), 도 8D는 GR 및 β-actin의 상대적인 발현 비율을 덴시토미트리(densitometry)를 이용하여 측정한 결과이고, 도 8E는 GSSG/GSH의 비율을 나타낸 결과이다.1 is a hearing test result obtained by ALA pretreatment or ALA post-treatment and ABR measurement in cisplatin-injected mice (*p <0.05 vs cisplatin alone, control group; control group, cisplatin group; CP group, ALA pretreatment) Group; ALA pre-treatment group, ALA post-treatment group; ALA post-treatment group, ALA group; ALA group, ABR; auditory brainstem response).
FIG. 2 is a result of histological analysis of the inner ear of cisplatin-injected mice subjected to ALA pretreatment or ALA post treatment (n = 3), and FIG. 2A shows the basal turn of the inner ear of the control mouse. 2B is an observation of the stria vascularis [top] and the spiral ganglion [bottom] at high magnification (double arrow (↔) is the thickness of the vascular stria, and an asterisk (*) is a spiral ganglion It shows the degeneration of cells, and the scale bar is 50 μm), FIG. 2C is a result of quantitative comparison of the vascular striatal thickness (unit: μm), and FIG. 2D is the basal, middle, and apical This is the result of comparing the number of spiral ganglion cells between each group in turn (*p <0.05 vs cisplatin alone).
3 is a result of confirming the therapeutic effect before and after administration of ALA on the damage to auditory hair cells induced by cisplatin in cisplatin-injected mice.FIG. 3A shows internal hair cells to confirm the survival or degeneration of hair cells And Phalloidin staining (red) of the organ of Corti including external hair cells (white asterisk (*) indicates degenerative hair cells, scale bar is 50 μm), and Figure 3B shows the cochlea of each group. ) Is the result of histogram quantification of the number of internal and external hair cells in the basal, middle, and apical turns of (*p <0.05 vs cisplatin alone).
FIG. 4 is a result of confirming the effect of ALA treatment on cell viability in cisplatin-treated HEI-OC1 cells. FIG. 4A shows HEI-OC1 cells 0.5, 1, 2, 3 and 4 Incubation with mM ALA for 30 hours and cytotoxicity was measured by MTT assay.FIG. 4B shows HEI-OC1 cells treated with 30 μM cisplatin for 30 minutes and then treated with 0.5 to 4 mM ALA for 1 hour. This is the result of measuring cytotoxicity by MTT assay (*p <0.05 vs cisplatin alone).
5 is a result of confirming the effect of ALA on cell cycle arrest and apoptosis in cisplatin-treated HEI-OC1 cells, and FIG. 5A is a flow cytometry method (flow flow cytometry), and FIG. 5B is a result of comparing the sub-G0/G1 ratio between the CP-only treatment group and the ALA-only treatment group, and FIG. 5C is for detecting apoptotic cells. It is the result of TUNEL assay analysis (when observed with a fluorescence microscope, DNA-green, nuclear-blue, scale bar is 100 μm), Figure 5D shows caspase-3 expression in cells treated with cisplatin and ALA. It is a Western blot analysis result for confirming, and FIG. 5E is a result of measuring the caspase-3:β-actin ratio using densitometry (*p <0.05 vs cisplatin alone).
Figure 6 is a result showing the ROS scavenging ability of ALA in cisplatin-treated cells, Figure 6A is the result of measuring the intracellular ROS level (level) using a DCFH-DA probe (fluorescence intensity using a flow cytometer Detection), Figure 6B is a result of measuring the relative FACS fluorescence intensities (*p <0.05 vs cisplatin alone).
7 is a result of confirming the antiapoptotic effect of ALA on Bax-mediated apoptosis by cisplatin, and FIG. 7A is a loading control of β-actin and Bax Western Blot analysis result (n = 3 per lane), Figure 7B is the result of measuring the relative expression ratio of Bax and β-actin using densitometry, Figure 7C is a loading control (loading control) β Western blot analysis results of -actin and Bcl-2 (n = 3 per lane), Figure 7D is the result of measuring the relative expression ratio of Bcl-2 and β-actin using densitometry (* p <0.05 vs cisplatin alone).
8 is a result showing the expression levels of antioxidant enzymes GPx and GR in cells before or after ALA treatment, and FIG. 8A is a Western blot analysis result of β-actin and GPx, which are loading controls (n = 3 per lane), Figure 8B is a result of measuring the relative expression ratio of GR and β-actin using densitometry, Figure 8C is a Western blot analysis result of β-actin and GR as loading control And (n = 3 per lane), FIG. 8D is a result of measuring the relative expression ratios of GR and β-actin using densitometry, and FIG. 8E is a result showing the ratio of GSSG/GSH.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 알파-리포익산(alpha-lipoic acid; ALA)을 유효성분으로 함유하는 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for treating ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.
상기 조성물은 청각유모세포 사멸을 억제시킬 수 있다. The composition may inhibit the death of auditory hair cells.
또한, 상기 조성물은 청각유모세포에서 Bax 유전자 또는 단백질 발현은 감소시키고, Bcl-2 유전자 또는 단백질 발현은 증가시킴으로써 미토콘리아 기능장애로 인한 이독성 난청을 줄여줄 수 있다. In addition, the composition can reduce the expression of Bax gene or protein in auditory hair cells and increase the expression of Bcl-2 gene or protein, thereby reducing ototoxic hearing loss due to mitochondrial dysfunction.
상기 조성물은 GPx 유전자 또는 단백질 발현을 증가시킬 수 있으며, 이는 GR 역할을 도와줌으로써 환원된 형태의 환원글루타티온(GSH)가 정상수준으로 유지될 수 있도록 기여할 수 있다. The composition may increase the expression of the GPx gene or protein, and this may contribute to maintaining the reduced form of reduced glutathione (GSH) at a normal level by helping the GR role.
본 발명의 한 구체예에서, 상기 약학조성물은 통상적인 방법에 따라 주사제, 과립제, 산제, 정제, 환제, 캡슐제, 좌제, 겔, 현탁제, 유제, 점적제 또는 액제로이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다.In one embodiment of the present invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to a conventional method. Formulations of can be used.
본 발명의 다른 구체예에서, 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer solution, bacteriostatic agent, which are commonly used in the preparation of pharmaceutical compositions. It may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders, and lubricants.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용 액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
본 발명의 일실시예에 따르면, 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to an embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or It can be administered to the subject in a conventional manner via the intradermal route.
본 발명에 따른 유효성분의 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다. The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be divided into several times, thereby not limiting the scope of the present invention.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나,이들 예에 한정되는 것은 아니다.In the present invention, the'subject' may be a mammal including a human, but is not limited to these examples.
또한, 본 발명은 알파-리포익산(alpha-lipoic acid; ALA)을 유효성분으로 함유하는 시스플라틴에 의한 이독성 난청 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for improving ototoxic hearing loss by cisplatin containing alpha-lipoic acid (ALA) as an active ingredient.
상기 건강기능식품은 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, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다.The health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
그밖에 천연 과일 주스, 합성 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 건강기능식품 조성물은 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알코올 및 비타민 복합제 중 어느 하나의 형태일 수 있다.In addition, it may contain pulp for the manufacture of natural fruit juices, synthetic fruit juices and vegetable beverages. These components may be used independently or in combination. In addition, the health functional food composition is any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex Can be
또한, 상기 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합 여부는 다른 규정이 없는 한 식품의약품안전청에 승인된 식품첨가물공전의 총칙 및 일반 시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.In addition, the above health functional food may additionally contain food additives, and whether it is suitable as a "food additive" is determined according to the general rules and general test methods of the Food Additive Code approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the relevant standards and standards.
상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀룰로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류 첨가 알칼리제, 보존료제제, 타르색소 제제 등의 혼합 제제류 등을 들 수 있다.Items listed in the "Food Additives Code", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as reduced pigment, licorice extract, crystalline cellulose, high cooling pigment, guar gum, L -Mixed preparations, such as a sodium glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.
이때, 건강기능식품을 제조하는 과정에서 식품에 첨가되는 프라바스타틴 및 항혈소판제는 필요에 따라 그 함량을 적절히 가감할 수 있으며, 바람직하게는 식품 100 중량부에 1 중량부 내지 90 중량부 포함되도록 첨가될 수 있다.At this time, the content of the pravastatin and antiplatelet agents added to the food in the process of manufacturing the health functional food can be appropriately added or subtracted as needed, and preferably added to include 1 to 90 parts by weight to 100 parts by weight of the food. I can.
더불어, 본 발명은 알파-리포익산(alpha-lipoic acid)을 유효성분으로 포함하는, 인 비트로(in vitro)에서 Bax 단백질 발현을 억제하거나, Bcl-2 단백질 발현을 증가시키거나, 또는 GPx 단백질 발현을 증가시키는 시약용 조성물을 제공한다. In addition, the present invention inhibits Bax protein expression, increases Bcl-2 protein expression, or GPx protein expression in vitro , which contains alpha-lipoic acid as an active ingredient. It provides a composition for a reagent that increases.
또한, 인간을 제외한 동물에, 알파-리포익산(alpha-lipoic acid)을 유효성분으로 포함하는 조성물을 처리하는 것을 특징으로 하는, Bax 단백질 발현을 억제하거나, Bcl-2 단백질 발현을 증가시키거나, GPx 단백질 발현을 증가시키는 방법을 제공한다. 즉, 본 발명에 따른 알파-리포익산은 Bax 단백질 발현만 억제할 수도 있고, Bcl-2 단백질 발현만을 증가시킬 수도 있으며, GPx 단백질 발현만을 증가시킬 수도 있다.In addition, animals other than humans, characterized by treating a composition containing alpha-lipoic acid as an active ingredient, inhibiting Bax protein expression, or increasing Bcl-2 protein expression, Methods of increasing GPx protein expression are provided. That is, the alpha-lipoic acid according to the present invention may inhibit only Bax protein expression, may increase only Bcl-2 protein expression, or may increase only GPx protein expression.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
<실험 방법><Experiment method>
1) 마우스 준비1) mouse preparation
암컷 및 수컷 마우스(8 주령)는 Hyochang Science(대구, 대한민국)에서 구입하였다. 모든 동물 절차는 경북대학교 동물 연구 위원회(Committee of Animal Research)가 발행한 Institutional Animal Care 지침에 부합하게 수행하였다.Female and male mice (8 weeks old) were purchased from Hyochang Science (Daegu, Korea). All animal procedures were performed in accordance with the Institutional Animal Care guidelines issued by the Committee of Animal Research at Kyungpook National University.
2) 생체 내 실험 디자인(In vivo experimental design)2) In vivo experimental design
마우스를 대조군(control group), 시스플라틴 군(CP group), ALA 전처리 군(ALA pre-treatment group), ALA 후처리 군(ALA post-treatment group) 및 ALA 군(ALA group)으로 나누었다(총 5 군).Mice were divided into control group, cisplatin group (CP group), ALA pre-treatment group, ALA post-treatment group, and ALA group (a total of 5 groups). ).
대조군(control group)은 0.9% 식염수로 3 일 동안만 처리하였다.The control group was treated with 0.9% saline for only 3 days.
ALA 전처리 군(ALA pre-treatment group)은 마우스에 ALA를 2 일간 주사한 후 시스플라틴을 투여하였다.In the ALA pre-treatment group, mice were injected with ALA for 2 days and then cisplatin was administered.
ALA 후처리 군(ALA post-treatment group)은 마우스에 시스플라틴을 주사한 후 다음 2 일 동안 ALA를 주사하였다. In the ALA post-treatment group, mice were injected with cisplatin and then ALA was injected for the next 2 days.
ALA 군은 마우스에 3 일 동안 ALA만 주사하였다.The ALA group injected only ALA into mice for 3 days.
시스플라틴 군(CP group)은 마우스에 시스플라틴만 주사하였다.In the cisplatin group (CP group), only cisplatin was injected into mice.
구체적으로 사용량은 20 m/kg 시스플라틴(Sigma, St. Louis, MO, USA) 및/또는 100 mg/kg ALA(Thioctacid inj®, Bukwang Pharmaceutical Co., Ltd., Seoul, 한국)을 복강 내 투여하였다.Specifically, 20 m/kg cisplatin (Sigma, St. Louis, MO, USA) and/or 100 mg/kg ALA (Thioctacid inj®, Bukwang Pharmaceutical Co., Ltd., Seoul, Korea) were administered intraperitoneally. .
아울러, 시스플라틴을 투여 받은 그룹의 경우, 청력 능력의 변화를 평가하기 위해, ABR 역치는 주입 전과 시스플라틴 주입 후 4.5 일에 측정하였다.In addition, in the case of the cisplatin-administered group, the ABR threshold was measured before injection and 4.5 days after cisplatin injection in order to evaluate the change in hearing ability.
3) 청각의 뇌간 반응 측정(auditory brainstem response; ABR)3) Auditory brainstem response (ABR)
청각 기능을 평가하기 위해, ABR 측정기(System 3, Tucker Davis Technology, Inc., Alachua, FL, USA)를 사용하여 ABR 측정을 수행했다. 모든 테스트는 방음실에서 이루어졌다. ABR 측정에 앞서 alfaxalone(40 mg/kg)을 근육 내 주사하여 마우스를 마취시키고 체온을 37℃로 유지하기 위해 가열 패드 위에 마우스를 놓았다. 마우스 체온은 직장 온도계로 측정하고 모니터링 하였다. ABR을 기록하기 위해 피하 주사 전극을 정수리(+charge), 유양돌기(-e) 및 뒷다리(ground)에 삽입하였다. 음향 자극(Acoustic stimuli)은 8, 16, 및 32 kHz의 주파수, 또는 일시적인 클릭 자극(click stimuli)에서 1-ms rise/fall time 및 5-ms plateau를 갖는 톤-버스트 자극(tone-burst stimulus)으로 구성되며, 스피커를 통해 모노럴(monaural) 방식으로 적용하였다.To evaluate auditory function, ABR measurements were performed using an ABR meter (
자극 신호(stimulus signals)는 SigGenRP 및 an RP2.1 real-time processor를 사용하여 생성시킨 다음 감쇠기(PA5, TDT), 스피커 드라이버(ED1, TDT) 및 정전 스피커(EC1, TDT)를 통해 전송되었다. 자극은 90-dB 음압 레벨부터 음향 임계값(acoustic threshold)까지 5-dB 감소율(decrement)로 500 회 반복하여 생성하였다. Stimulus signals were generated using SigGenRP and an RP2.1 real-time processor and then transmitted through attenuators (PA5, TDT), speaker drivers (ED1, TDT) and electrostatic speakers (EC1, TDT). Stimulation was generated by repeating 500 times at a 5-dB decrement from the 90-dB sound pressure level to the acoustic threshold.
4) 파라핀 절편(Paraffin sectioning) 및 조직학적 분석(histological analysis)4) Paraffin sectioning and histological analysis
마우스를 1X 인산염 완충 식염수(PBS)로 관류시키고, 내이(inner ears)를 분리하였다. 파라핀 절편(Paraffin sectioning)의 경우 4℃에서 24 시간 동안 4% 파라포름알데히드(PFA)가 있는 PBS안에 내이를 고정시킨 후, 4℃에서 24 시간 동안 10% 에틸렌디아민테트라아세트산(ethylenediaminetetraacetic acid)이 있는 PBS로 탈석회화(decalcify)시켰다. 안쪽 귀(inner ears)를 에탄올 시리즈(graded ethanol series)로 탈수시키고, 자일렌(xylene)을 침투시키고, 실온에서 파라핀에 매립시켰다.Mice were perfused with 1X phosphate buffered saline (PBS), and inner ears were separated. In the case of paraffin sectioning, the inner ear was fixed in PBS containing 4% paraformaldehyde (PFA) for 24 hours at 4°C, and then 10% ethylenediaminetetraacetic acid was added at 4°C for 24 hours. It was decalcified with PBS. The inner ears were dehydrated with a graded ethanol series, xylene was permeated, and embedded in paraffin at room temperature.
파라핀으로 봉입된 내이(paraffin-embedded inner ears)는 마이크로톰(microtome)(Leica RM2235, Leica Microsystems, Germany)을 사용하여 6 μm 두께의 조각으로 연속적으로 절편을 만들고, 염색을 위해 Superfrost Plus® 현미경 슬라이드(Fisher Scientific, Pittsburgh, PA, USA)에 올렸다. 모든 슬라이드는 사용할 때까지 4℃로 유지하였다. 파라핀으로 봉입된 내이를 가진 슬라이드를 65℃에서 1 시간 동안 배양하고 자일렌(xylene)으로 탈파라핀화한 후 에탄올 시리즈(graded ethanol series)를 사용하여 재수화(rehydrattion)시켰다. 핵(Nuclei) 및 세포질(cytoplasm)은 헤마톡실린 및 에오신 시약(Sigma)으로 염색되었다. Paraffin-embedded inner ears were successively sectioned into 6 μm-thick pieces using a microtome (Leica RM2235, Leica Microsystems, Germany), and a Superfrost Plus® microscope slide ( Fisher Scientific, Pittsburgh, PA, USA). All slides were kept at 4°C until use. A slide with an inner ear encapsulated with paraffin was incubated at 65° C. for 1 hour, deparaffinized with xylene, and then rehydrated using a graded ethanol series. Nuclei and cytoplasm were stained with hematoxylin and eosin reagents (Sigma).
나선형 신경절 세포(spiral ganglion cells)의 밀도를 확인하기 위해, 달팽이관(cochlea)의 각 회전(turn)[기저(basal), 중간(middle) 및 정점(apical)]에서, 파라핀 섹션의 1 mm2 영역 내 나선형 신경절 세포의 평균수(average number)를 계산하여 세포의 비율을 계산하였다.To determine the density of spiral ganglion cells, in each turn (basal, middle and apical) of the cochlea, 1 mm 2 area of the paraffin section. The percentage of cells was calculated by calculating the average number of inner spiral ganglion cells.
5) 팔로이딘 염색(Phalloidin staining)5) Phalloidin staining
달팽이관(cochlea)을 4% PFA가 포함된 PBS로 실온에서 20 분간 고정하고, 1X PBS로 세척하며, 0.1% Triton X-100로 실온에서 15 분 동안 항온 배양하였다. 달팽이관으로부터 코르티 기관을 해부하여, 암실에서 2 시간 동안 Alexa Fluor 555 phalloidin(Invitrogen-Molecular Probes, Eugene, OR, USA, 1 : 1000)이 포함된 PBS로 염색하고, PBS로 3 번 세척하였다. 코르티 기관을 공초점 현미경(confocal microscope, LSM 700, Zeiss, Oberkochen, Germany)을 이용하여 홀 마운트(whole mounts) 관찰하여 달팽이관의 각 회전(turn)의 200 μm 영역 내에서 정상 유모세포(normal hair cells)의 수를 세었다.The cochlea was fixed with PBS containing 4% PFA for 20 minutes at room temperature, washed with 1X PBS, and incubated with 0.1% Triton X-100 for 15 minutes at room temperature. The organ of Corti was dissected from the cochlea, stained with PBS containing Alexa Fluor 555 phalloidin (Invitrogen-Molecular Probes, Eugene, OR, 1:1000) for 2 hours in the dark, and washed 3 times with PBS. Using a confocal microscope (LSM 700, Zeiss, Oberkochen, Germany) to observe the corti organs with hole mounts, normal hair cells within the 200 μm area of each turn of the cochlea. ) Counted.
6) 세포 배양 및 생존력 분석(Cell culture and viability assay)6) Cell culture and viability assay
HEI-OC1 청각유모세포를 항생제가 없는 10% 태아 소 혈청(Hyclone, Logan, UT, USA)을 함유한 Dulbecco's modified Eagle's medium(Hyclone, Logan, UT, USA) 및 50 units/mL IFN-γ(PeproTech, EC, London, UK)내에서 16 시간 동안 허용 조건(33℃, 10% CO2) 아래에서 배양하였다. HEI-OC1 auditory hair cells were treated with antibiotic-free 10% fetal bovine serum (Hyclone, Logan, UT, USA) in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA) and 50 units/mL IFN-γ (PeproTech). , EC, London, UK) for 16 hours under acceptable conditions (33° C., 10% CO 2 ).
시험관 내 모든 실험에서 30 시간 동안 30 μM 시스플라틴 처리 전후에 각각의 세포 그룹(7 × 103 cells/well of 96-well plate)을 1 mM ALA로 전 처리 또는 사후 처리하였다. In all experiments in vitro, each group of cells (7 × 10 3 cells/well of 96-well plate) was pre-treated or post-treated with 1 mM ALA before and after 30 μM cisplatin treatment for 30 hours.
세포 생존력은 3-(4,5-디메틸티아졸-2-yl)-2,5-디페닐테트라졸리움 브로마이드[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT; Sigma, St. Louis, MO, USA)]를 사용하여 측정하였다. 세포를 0.5, 1, 2, 3, 4 mM의 ALA 및/또는 30 μM 시스플라틴으로 30 시간 동안 처리한 후, 0.5 mg /mL의 MTT 용액을 세포 배양 배지에 첨가한 후, 33℃에서 10% CO2와 함께 2 시간 동안 배양하였다. 각 웰의 광학 밀도(optical density)를 550 nm에서 마이크로플레이트 판독기(microplate reader)를 사용하여 측정하였다.Cell viability is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide( MTT; Sigma, St. Louis, MO, USA)]. After the cells were treated with 0.5, 1, 2, 3, 4 mM ALA and/or 30 μM cisplatin for 30 hours, 0.5 mg/mL of MTT solution was added to the cell culture medium, followed by 10% CO at 33°C. with 2 and cultured for 2 hours. The optical density of each well was measured at 550 nm using a microplate reader.
7) 세포 내 ROS 생산 측정(Measurement of intracellular ROS production)7) Measurement of intracellular ROS production
HEI-OC1 세포를 16 시간 동안 배양한 다음, ALA 전후 처리의 존재 또는 부재 하에 24 시간 동안 30 μM 시스플라틴으로 처리하였다. 형광 염료 인 DCFH-DA(Invitrogen-Molecular Probes, Eugene, OR, USA)를 사용하여 세포 내 ROS 수준을 측정하였다. 세포를 1 μM PBS로 2 회 세척하고 10 μM DCF-DA로 33℃, 10% CO2 조건에서 5 분간 배양하였다. 그런 다음, BD FACS Aria III 유세포분석기(BD Biosciences, San Diego, CA, USA)를 사용하여 DCFH-DA에서 배양된 세포의 유세 분석을 수행하였다(10,000 events per sample).HEI-OC1 cells were incubated for 16 hours and then treated with 30 μM cisplatin for 24 hours with or without ALA pre- and post-treatment. The intracellular ROS level was measured using a fluorescent dye DCFH-DA (Invitrogen-Molecular Probes, Eugene, OR, USA). The cells were washed twice with 1 μM PBS and incubated for 5 minutes at 33° C. and 10% CO 2 with 10 μM DCF-DA. Then, flow analysis of cells cultured in DCFH-DA was performed using a BD FACS Aria III flow cytometer (BD Biosciences, San Diego, CA, USA) (10,000 events per sample).
8) 세포주기 분석(Cell cycle analysis)8) Cell cycle analysis
HEI-OC1 세포에 트립신을 처리하고, 수를 세고, 원심분리 하고, 30 μM 시스플라틴를 처리한 후 30 시간 동안 70% 에탄올에 고정시켰다. HEI-OC1 세포를 1X PBS로 2 회 세척하고 원심분리 시켰다. 원심분리된 펠렛을 RNase A (0.2 mg/mL) 및 프로피디움 요오드화물(PI, 10 μg / mL)의 용액에 재현탁 시키고 4℃에서 30 분 동안 항온 배양하였다. 세포주기 분포(Cell cycle distribution)는 BD FACS Aria III 유세포분석기(BD Biosciences, San Diego, CA, USA)를 사용하여 측정하였다.HEI-OC1 cells were trypsinized, counted, centrifuged, treated with 30 μM cisplatin, and fixed in 70% ethanol for 30 hours. HEI-OC1 cells were washed twice with 1X PBS and centrifuged. The centrifuged pellet was resuspended in a solution of RNase A (0.2 mg/mL) and propidium iodide (PI, 10 μg/mL) and incubated at 4° C. for 30 minutes. Cell cycle distribution was measured using a BD FACS Aria III flow cytometer (BD Biosciences, San Diego, CA, USA).
9) DNA 단편의 검출(Detection of DNA fragmentation)9) Detection of DNA fragmentation
아파토시스(apoptosis)는 terminal TUNEL technique에 기초로 in situ 세포 사멸 검출 키트(Roche Biochemicals, Mannheim, Germany)를 사용하여 분석하였다. HEI-OC1 세포를 24-웰 배양 플레이트 상에서 배양하고, 시스플라틴으로 30 시간 동안 처리하였다. 그런 다음 각 실험 그룹의 세포를 실온에서 15 분 동안 4% PFA에 고정시킨 후 세척하고, 0.1% Triton X-100 및 0.1% 시트르산 나트륨이 포함된 물로 4℃에서 5 분간 침투(permeabilize)시켰다. TUNEL 반응 혼합물 50 μL을 샘플에 첨가하고 37℃에서 60 분 동안 항온 처리하였다. 모든 샘플을 각각 5 분 동안 1X PBS로 3 회 세척하였다. 그런 다음, 핵을 4',6-디아미디노-2-페닐인돌(4′, 6-diamidino-2-phenylindole)로 5 분 동안 염색시켰다. 형광현미경(fluorescence microscopy, Carl Zeiss, Oberkochen, Germany)을 사용하여 시편을 시각화하였다.Apatosis was analyzed using an in situ cell death detection kit (Roche Biochemicals, Mannheim, Germany) based on the terminal TUNEL technique. HEI-OC1 cells were cultured on a 24-well culture plate and treated with cisplatin for 30 hours. Then, the cells of each experimental group were fixed in 4% PFA at room temperature for 15 minutes, washed, and permeabilized at 4° C. for 5 minutes with water containing 0.1% Triton X-100 and 0.1% sodium citrate. 50 μL of the TUNEL reaction mixture was added to the sample and incubated at 37° C. for 60 minutes. All samples were washed 3 times with 1X PBS for 5 minutes each. Then, the nuclei were stained with 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole) for 5 minutes. Specimens were visualized using a fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).
10) 단백질 준비(Protein preparation)10) Protein preparation
HEI-OC1 세포를 얼음처럼 차가운 1X PBS로 세척하고 프로테아제 억제제 (Calbiochem Protease Inhibitor Cocktail Set 1, La Jolla, CA, USA)의 칵테일(cocktail)이 들어있는 RIPA 완충액(Elpis Biotech, Daejeon, Korea) 100μL에 현탁시켰다. 이러한 현탁액을 미리 냉각시킨 1.5 mL 튜브에 옮기고 얼음에서 15 분 동안 배양한 다음 4℃에서 13,000 rpm으로 30 분간 원심분리하고 1 분 동안 볼텍싱 하였다. 상등액을 취하여 얼음 위의 새 튜브 안에 넣고 펠릿은 버렸다.HEI-OC1 cells were washed with ice-cold 1X PBS, and in 100 μL of RIPA buffer (Elpis Biotech, Daejeon, Korea) containing a cocktail of protease inhibitor (Calbiochem Protease Inhibitor Cocktail Set 1, La Jolla, CA, USA). Suspended. This suspension was transferred to a pre-cooled 1.5 mL tube, incubated on ice for 15 minutes, centrifuged at 13,000 rpm at 4° C. for 30 minutes, and vortexed for 1 minute. The supernatant was taken and placed in a new tube on ice and the pellet was discarded.
11) 웨스턴 블롯 분석(Western blot analysis)11) Western blot analysis
HEI-OC1 세포 추출물을 12% 소듐 도데실 설페이트-폴리아크릴아미드 겔 전기영동으로 분석하였다. 분리된 단백질을 니트로 셀룰로오스 막(nitrocellulose membrane)에 전기 전이시켰다. 막을 5% 탈지유를 함유한 20 mM Tris-HCl(pH 7.6), 137 mM NaCl 및 0.01% Tween-20(TBS-T)의 용액으로 1 시간 동안 블럭(block)한 후, 1차 항체(1 : 100-1 : 2000 dilution)로 실온에서 탐침(probe)시켰다. 다음으로, 막(membrane)을 TBS-T로 각각 5 분 동안 3 회 세척하고, 2차 항체(1 : 2000 dilution)을 배양시켰다. 일련의 세척 과정을 진행한 후에, 향상된 화학발광검출시스템(chemiluminescent detection system)을 사용하여 측정하였다.The HEI-OC1 cell extract was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated protein was electrically transferred to a nitrocellulose membrane. After blocking the membrane for 1 hour with a solution of 20 mM Tris-HCl (pH 7.6), 137 mM NaCl and 0.01% Tween-20 (TBS-T) containing 5% skim milk, the primary antibody (1: 100-1: 2000 dilution) was probed at room temperature. Next, the membrane (membrane) was washed three times for 5 minutes each with TBS-T, and a secondary antibody (1: 2000 dilution) was incubated. After undergoing a series of washing procedures, measurements were made using an improved chemiluminescent detection system.
Rabbit anti-Bax 및 anti-caspase-3 항체는 Cell Signaling(Cell Signaling, Danvers, MA, USA), rabbit anti-Bcl-2, anti-GR, 및 anti-GPx 항체는 Abcam(Abcam, Cambridge, MA, UK)부터 구입하였다. 그리고 Goat anti-rabbit-lgG-HRP를 2 차 항체로 사용하였다(Cell Signaling, Danvers, MA, USA).Rabbit anti-Bax and anti-caspase-3 antibodies are Cell Signaling (Cell Signaling, Danvers, MA, USA), rabbit anti-Bcl-2, anti-GR, and anti-GPx antibodies are Abcam (Abcam, Cambridge, MA, UK). And Goat anti-rabbit-lgG-HRP was used as a secondary antibody (Cell Signaling, Danvers, MA, USA).
12) 글루타티온 수치 측정(Measuring glutathione levels)12) Measuring glutathione levels
GSH 수치(Cellular GSH levels)는 24 시간 동안 시스플라틴에 노출된 HEI-OC1 세포 용해물을 GSH colorimetric detection kit(BioVision Inc., Milpitas, CA, USA)를 사용하여 측정하였다. 간단히 말하면, 세포를 긁어서 얼음으로 차갑게 된 글루타티온 완충액 60 μL에 현탁시키고, 각 샘플을 과염소산 20 μL와 혼합하였다. 샘플을 얼음 상에서 5 분간 유지하고, 4℃에서 2 분 동안 13,000 rpm에서 원심 분리하였다. 상등액을 수집하고, 40 μL의 생성된 샘플에 20 μL의 차가운 6 N KOH 용액을 첨가하였다. 이들 샘플을 얼음 위에서 5 분간 유지하고 추운 곳에서 원심분리 하였다. 다음으로, 10 μL의 상등액(neutralized supernatant)을 96-웰 플레이트로 옮겼다. GSH를 검출하기 위해, 80 μL 분석 완충액을 각 웰에 첨가하였다. 총 GSH 샘플 70 μL을 10 μL reducing agent mix에 첨가하고 10 분간 배양하였다. GSSG 샘플을 60 μL 분석 완충액 및 10 μL GSH quencher에 10 분 동안 첨가한 다음, 10 μL 환원제(reducing agent)를 첨가하고, GSSG 샘플을 10 분 동안 배양하였다. 마지막으로, 10 μL의 σ-프탈알데히드(o-phthalaldehyde)를 모든 샘플에 첨가하고 실온에서 40 분 동안 배양하였다.Cellular GSH levels were measured using a GSH colorimetric detection kit (BioVision Inc., Milpitas, CA, USA) of HEI-OC1 cell lysates exposed to cisplatin for 24 hours. Briefly, cells were scraped and suspended in 60 μL of ice-cold glutathione buffer, and each sample was mixed with 20 μL of perchloric acid. Samples were kept on ice for 5 minutes and centrifuged at 13,000 rpm for 2 minutes at 4°C. The supernatant was collected and 20 μL of cold 6 N KOH solution was added to 40 μL of the resulting sample. These samples were kept on ice for 5 minutes and centrifuged in a cold place. Next, 10 μL of the supernatant (neutralized supernatant) was transferred to a 96-well plate. To detect GSH, 80 μL assay buffer was added to each well. 70 μL of a total GSH sample was added to 10 μL reducing agent mix and incubated for 10 minutes. GSSG samples were added to 60 μL assay buffer and 10 μL GSH quencher for 10 minutes, then 10 μL reducing agent was added, and GSSG samples were incubated for 10 minutes. Finally, 10 μL of σ-phthalaldehyde (o-phthalaldehyde) was added to all samples and incubated at room temperature for 40 minutes.
13) 통계분석(Statistical analyses)13) Statistical analyses
SPSS 통계 소프트웨어 패키지(version 23; SPSS, Chicago, IL, USA)를 사용하여 데이터를 분석하였다. 연속 변수(Continuous variables)는 평균±표준 편차(mean±standard deviation)로 나타내었다. One-way ANOVA(일원분산분석)을 사용하여 5 개 또는 6 개 그룹의 변수를 분석하였다. Student 's t test를 사용하여 두 그룹을 분석하였다. 0.05 미만의 p 값(P<0.05)은 통계적으로 유의하다고 간주되었다.Data were analyzed using the SPSS statistical software package (version 23; SPSS, Chicago, IL, USA). Continuous variables were expressed as mean±standard deviation. One-way ANOVA (one-way analysis of variance) was used to analyze 5 or 6 groups of variables. Two groups were analyzed using Student's t test. P values less than 0.05 (P<0.05) were considered statistically significant.
<실시예 1> ALA의 청력 보호 및 시스플라틴 유도 이독성에 의한 감각세포 사멸 억제 효과 확인<Example 1> Confirmation of the effect of ALA to protect hearing and inhibit sensory cell death by cisplatin-induced ototoxic
1) 청각 뇌간 반응(auditory brainstem response) 측정을 통한 확인1) Confirmation through measurement of auditory brainstem response
시스플라틴에 의해 유발된 이독성 난청에 ALA가 미치는 영향을 마우스 모델을 통해 조사하였다. 대조군(control group)과 ALA 군(ALA group)의 경우, ABR 임계값의 변화는 클릭 음을 포함한 전체 주파수 범위에서 5 dB 미만이었다 라는 점에서, 정상 마우스에서 ALA 처리가 청각 능력에 영향을 미치지 않는다는 것을 알 수 있었다. 또한, 시스플라틴 군(CP group)은 ABR 역치의 유의한 변화를 나타내어 시스플라틴 치료가 심각한 난청을 유발한다는 것을 확인하였다(도 1).The effect of ALA on cisplatin-induced ototoxic hearing loss was investigated through a mouse model. In the case of the control group and the ALA group, the change in the ABR threshold was less than 5 dB over the entire frequency range including the click sound, indicating that ALA treatment did not affect hearing ability in normal mice. I could see that. In addition, the cisplatin group (CP group) showed a significant change in the ABR threshold, confirming that cisplatin treatment caused severe hearing loss (FIG. 1).
ALA 전처리 군(ALA pre-treatment group)은 청력이 거의 완벽할 정도로 보호되었다는 것을 통해(ABR 임계값에서 5-10 dB 변화), ALA가 시스플라틴에 의한 청력 상실을 보호할 수 있다는 점을 입증하게 되었다. 가장 흥미로운 결과는 ALA 후처리 군(ALA post-treatment group)에 의해 알 수 있듯이 ALA의 경감 효과(alleviative effect)였다. ALA 전처리 군(ALA pre-treatment group)과 ALA 후처리 군(ALA post-treatment group)에 ABR 역치 변화가 5-10 dB로 작은 차이가 관찰되었지만, ALA 후처리 군(ALA post-treatment group)에서 시스플라틴 투여 후 대부분 청력 감소를 완화시켰다. 이러한 결과는 ALA가 치료 약물로서 효과가 있음을 시사한다.The ALA pre-treatment group demonstrated that hearing was almost completely protected (5-10 dB change in ABR threshold), demonstrating that ALA can protect against cisplatin-induced hearing loss. . The most interesting result was the alleviative effect of ALA, as can be seen by the ALA post-treatment group. A small difference in ABR threshold change of 5-10 dB was observed between ALA pre-treatment group and ALA post-treatment group, but in ALA post-treatment group. Most of the hearing loss was alleviated after cisplatin administration. These results suggest that ALA is effective as a therapeutic drug.
2) 혈관선조(stria vascularis) 및 나선형 신경절(spiral ganglion)의 형태학적 관찰을 통한 확인2) Confirmation through morphological observation of stria vascularis and spiral ganglion
듣기(hearing) 능력의 변화가 형태학적 이상과 관련이 있는지를 알아보기 위해 헤마톡실린(hematoxylin)과 에오신(eosin) 염색을 이용한 조직학적 분석을 수행하였다. 혈관선조(stria vascularis)와 나선형 신경절(spiral ganglion)이 시스플라틴로 인한 이독성의 주요 표적이기 때문에 이 두 영역에 중점을 두고 조사하였다(도 2A).Histological analysis using hematoxylin and eosin staining was performed to determine whether changes in listening ability were related to morphological abnormalities. Since the stria vascularis and the spiral ganglion are the main targets of ototoxicity due to cisplatin, the study focused on these two areas (Fig. 2A).
혈관선조(stria vascularis)에서는 대조군과 다른 모든 군 사이의 두께를 포함하여 두드러진 조직학적 차이는 관찰되지 않았다(도 2B (위) 및 도 2C). 시스플라틴에 의한 가장 심한 손상은 나선형 신경절(spiral ganglion)에서 발견되었는데, 세포수가 대조군(control group) 보다 시스플라틴 군(CP group)의 basin turn에서 70% 더 낮았다. 대조적으로, ALA 전처리 군(ALA pre-treatment group) 및 ALA 후처리 군(ALA post-treatment group)에서 나선형 신경절 세포수는 대조군과 유사하였다(도 2B (아래) 및 도 2D).In the stria vascularis, no significant histological differences were observed, including the thickness between the control group and all other groups (FIGS. 2B (top) and FIG. 2C). The most severe damage caused by cisplatin was found in the spiral ganglion, and the number of cells was 70% lower in the basin turn of the cisplatin group (CP group) than in the control group. In contrast, in the ALA pre-treatment group and the ALA post-treatment group, the number of spiral ganglion cells was similar to that of the control group (FIGS. 2B (bottom) and FIG. 2D).
이러한 결과는 나선형 신경절 세포의 손실이 시스플라틴으로부터 직접 난청을 유발시킬 수 있음을 시사하는 것이며, 이는 ABR 임계값 결과와 매우 일치함을 확인할 수 있었다.These results suggest that the loss of helical ganglion cells can induce hearing loss directly from cisplatin, which is in good agreement with the ABR threshold result.
3) 유모세포(hair cell)의 조직학적 변화 관찰을 통한 확인3) Confirmation through observation of histological changes in hair cells
유모세포(hair cell)가 시스플라틴에 의한 이독성 난청에 영향을 받는지 여부를 확인하기 위해 코르티(corti) 기관의 홀 마운트 팔로이딘(whole mount phalloidin) 염색을 하여 조사하였다. 그 결과, 시스플라틴 투여로 인한 내부 유모세포와 외부 유모세포의 현저한 변화를 확인할 수 있었다(도 3).To determine whether hair cells are affected by cisplatin-induced ototoxic hearing loss, corti organs were stained with whole mount phalloidin. As a result, remarkable changes in internal hair cells and external hair cells due to the administration of cisplatin could be confirmed (FIG. 3).
기저부 회전(basal turn)의 면적은 중간 회전(middle turn) 및 정점 회전(apical turn)의 영역보다 더 심한 손상을 보였으며, 바깥쪽 유모 세포(outer hair cell)는 안쪽 유모 세포(inner hair cell)보다 시스플라틴에 더 민감하였다(도 3A). 그러나 심각한 손상은 ALA로 전처리 또는 후처리함으로써 현저하게 보호(protect)되거나 경감(alleviate)된 것을 확인할 수 있었다(도 3B).The area of the basal turn showed more severe damage than the area of the middle and apical turns, and the outer hair cells were inner hair cells. It was more sensitive to cisplatin (Fig. 3A). However, it was confirmed that severe damage was significantly protected or alleviated by pretreatment or post-treatment with ALA (Fig. 3B).
<실시예 2> ALA의 세포 독성 및 세포 생존율 확인<Example 2> Confirmation of cytotoxicity and cell viability of ALA
시스플라틴에 의해 유도된 세포 독성에 ALA가 영향을 미치는지 알아보기 위해 HEI-OC1(House Ear Institute-Organ of Corti 1) 세포를 사용하여 ALA가 조절하는 세포 내 메커니즘을 조사하고자 하였다. 이에 먼저 ALA가 처리되지 않은 세포와 ALA 처리된 세포의 세포생존력을 측정하여 ALA의 세포 독성을 시험했다.To investigate whether ALA affects the cytotoxicity induced by cisplatin, we used HEI-OC1 (House Ear Institute-Organ of Corti 1) cells to investigate the intracellular mechanisms regulated by ALA. First, the cytotoxicity of ALA was tested by measuring the viability of cells not treated with ALA and cells treated with ALA.
0.5 ~ 4 mM 농도의 ALA에서 세포 독성이 없음을 관찰할 수 있었다(도 4A). 단, 시스플라틴만 처리된 경우(시스플라틴 군(CP group)) 세포 생존율을 40% 까지 감소하였다. 그러나, ALA의 전처리 또는 사후처리로, 세포 생존 능력은 유의하게 증가되었음을 확인할 수 있었다(도 4B). 세포에 2 mM ALA가 전처리 되었을 때 가장 높은 세포 생존률인 87%의 수치가 확인되었다. 또한, ALA가 후처리 되었을 때 시스플라틴에 의한 세포 손상을 상당히 완화되어 1 mM ALA에서 78%의 세포 생존률이 확인되었다. 이러한 결과를 고려하여, 다음 실험에서 1 mM ALA를 사용했다.It could be observed that there was no cytotoxicity in ALA at a concentration of 0.5 to 4 mM (Fig. 4A). However, when only cisplatin was treated (cisplatin group (CP group)), the cell viability was reduced to 40%. However, with ALA pretreatment or post treatment, it could be confirmed that the cell viability was significantly increased (Fig. 4B). When the cells were pretreated with 2 mM ALA, the highest cell viability, 87%, was confirmed. In addition, when ALA was post-treated, cell damage caused by cisplatin was significantly alleviated, and a cell viability of 78% was confirmed in 1 mM ALA. In consideration of these results, 1 mM ALA was used in the next experiment.
<실시예 3> ALA의 세포 주기(cell cycle)와 세포사멸(apoptosis)에 미치는 영향 확인<Example 3> Confirmation of the effect of ALA on the cell cycle and apoptosis
시스플라틴의 세포사멸(apoptosis)에 대한 ALA의 방어적인 역할을 입증하기 위해 세포 주기(cell cycle)와 세포사멸 신호 전달 경로(apoptotic signaling pathway)를 분석하여 조사하였다.To demonstrate the protective role of ALA on cisplatin apoptosis, the cell cycle and apoptosis signaling pathway were analyzed and investigated.
종래의 연구는 시스플라틴과 DNA의 교차 결합이 세포 주기 억류(cell cycle arrest)를 일으킨다고만 제안했기 때문에, 본 발명에서는 유세포분석(flow cytometry)을 통해 sub-G0/G1 fraciton ratio를 측정하였다.Since conventional studies have only suggested that cross-linking of cisplatin and DNA causes cell cycle arrest, in the present invention, the sub-G0/G1 fraciton ratio was measured through flow cytometry.
시스플라틴 군(CP group)이 대조군(control group)보다 sub-G0/G1 단계에서 더 큰 세포 집단을 가지고 있음을 확인하였다(17% vs. 4%). 그러나, ALA 전처리 군(ALA pre-treatment group), ALA 후처리 군(ALA post-treatment group)은 세포 집단 중 6-7%만이 sub-G0/G1 단계에 있었다(도 5A 및 도 5B). 이러한 결과는 ALA가 세포를 보호하고 시스플라틴에 의해 유도된 세포 주기 억류(cell cycle arrest)를 완화시킬 수 있음을 시사한다.It was confirmed that the cisplatin group (CP group) had a larger cell population at the sub-G0/G1 stage than the control group (17% vs. 4%). However, in the ALA pre-treatment group and ALA post-treatment group, only 6-7% of the cell population was in the sub-G0/G1 stage (FIGS. 5A and 5B ). These results suggest that ALA can protect cells and mitigate cisplatin-induced cell cycle arrest.
시스플라틴 군(CP group)은 TUNEL assay 분석 시 대조군(control group)에서 보이지 않는 높은 TUNEL signal를 보였다. 대조적으로, ALA 전처리 군(ALA pre-treatment group)과 ALA 후처리 군(ALA post-treatment group)에서 최소 TUNEL 신호만 검출되어 시스플라틴 군(CP group) 보다 신호 강도가 현저히 낮았다(도 5C). The cisplatin group (CP group) showed a high TUNEL signal that was not seen in the control group when analyzed by the TUNEL assay. In contrast, only the minimum TUNEL signal was detected in the ALA pre-treatment group and the ALA post-treatment group, and the signal intensity was significantly lower than that of the cisplatin group (CP group) (Fig. 5C).
또한, caspase-3 발현 분석을 통해 ALA가 세포 사멸 신호를 감소시키는 능력이 있음을 웨스턴 블롯 분석을 통해 관찰할 수 있었다. 시스플라틴이 처리된 세포에서 caspase-3 발현이 많이 됨을 확인할 수 있었으나, 시스플라틴 처리되지 않은 대조군에서는 눈에 띄지 않을 만큼 발현되지 않았다. 대조적으로, ALA 전처리 군(ALA pre-treatment group) 및 ALA 후처리 군(ALA post-treatment group)의 경우 caspase-3 발현이 억제되는 것으로 관찰되었다(도 5D 및 도 5E).In addition, it was observed through Western blot analysis that ALA has the ability to reduce apoptosis signals through caspase-3 expression analysis. It was confirmed that caspase-3 expression was high in the cisplatin-treated cells, but the cisplatin-treated control group did not express enough to be conspicuous. In contrast, it was observed that caspase-3 expression was suppressed in the ALA pre-treatment group and ALA post-treatment group (FIGS. 5D and 5E).
이러한 결과는 ALA를 이용한 전처리가 시스플라틴 세포 독성에 대해 세포를 보호할 수 있을 뿐만 아니라, ALA를 이용한 후처리가 시스플라틴에 의해 유도된 caspase-3-의존성 세포사멸 시그널(caspase-3-dependent apoptosis signaling pathway)을 불활성화하여 세포 사멸을 완화시킬 수 있음을 의미한다.These results show that not only the pretreatment with ALA can protect cells against cisplatin cytotoxicity, but the post-treatment with ALA is the caspase-3-dependent apoptosis signaling pathway induced by cisplatin. ), which means that cell death can be alleviated.
<실시예 4> 미토콘드리아 내 ROS 감소 효과 및 관련 단백질의 증감 확인<Example 4> Confirmation of the effect of reducing ROS in mitochondria and increase or decrease of related proteins
시스플라틴에 의한 세포사멸(apoptosis)의 주요 원인 중 하나는 세포 내 ROS의 과도한 축적이며, ALA는 효과적인 항산화제로서 잘 알려져 있다. 이에, HEI-OC1 세포에서 ALA가 시스플라틴에 의해 생성된 세포 내 ROS를 감소시키는지를 확인하기 위해 DCFHDA(2′, 7′-dichlorofluorescein diacetate) 분석을 수행하였다. One of the main causes of cisplatin-induced apoptosis is excessive accumulation of ROS in cells, and ALA is well known as an effective antioxidant. Accordingly, DCFHDA (2′, 7′-dichlorofluorescein diacetate) analysis was performed to confirm whether ALA reduces intracellular ROS produced by cisplatin in HEI-OC1 cells.
시스플라틴 군(CP group)은 대조군(control group)에 비해 약 1.6 배의 형광 강도를 나타내었으며, 이는 ROS의 증가는 시스플라틴에 의한 것임을 알 수 있다. 대조적으로, ALA 전처리 군(ALA pre-treatment group)은 대조군(control group)과 유사한 형광 강도(fluorescent intensity)를 유지했다. 또한, ALA 후처리 군(ALA post-treatment group)은 시스플라틴 군(CP group)에 비해 현저히 낮은 형광 강도를 나타냈다(도 6A 및 도 6B). 이러한 결과는 ALA가 시스플라틴 처리 후 세포 내 과발현된 ROS를 감소시킬 뿐만 아니라, 처리 전에도 시스플라틴 유발된 ROS 생성을 예방한다는 것을 의미한다.The cisplatin group (CP group) showed about 1.6 times the fluorescence intensity compared to the control group (control group), which indicates that the increase in ROS was caused by cisplatin. In contrast, the ALA pre-treatment group maintained a similar fluorescent intensity as the control group. In addition, the ALA post-treatment group showed significantly lower fluorescence intensity compared to the cisplatin group (CP group) (FIGS. 6A and 6B). These results indicate that ALA not only reduces overexpressed ROS in cells after cisplatin treatment, but also prevents cisplatin-induced ROS production before treatment.
세포질에 존재하는 Bax는 산화 스트레스(oxidative stress)에 따라 미토콘드리아 막간 공간(mitochondrial intermembrane space)으로 이동하여 시토크롬 c를 방출함으로써 세포자멸(apoptosis)을 유도한다. 반면 Bcl-2는 Bax 활성을 억제하여 세포 사멸(apoptosis)을 억제하고 세포 생존을 촉진한다. 따라서 시스플라틴에 의해 증가된 ROS 축적은 Bax가 세포 사멸을 유도하는 미토콘드리아 기능 장애를 일으키도록 유도하는 것으로, ALA는 시스플라틴에 의해 유도된 산화 스트레스로부터 미토콘드리아를 보호하는데 중심적인 역할을 한다.Bax present in the cytoplasm moves to the mitochondrial intermembrane space according to oxidative stress and releases cytochrome c, thereby inducing apoptosis. On the other hand, Bcl-2 inhibits Bax activity, inhibits apoptosis and promotes cell survival. Thus, increased ROS accumulation by cisplatin induces Bax to cause mitochondrial dysfunction that induces cell death, and ALA plays a central role in protecting mitochondria from oxidative stress induced by cisplatin.
ALA의 ROS 스캐밴징(scavenging)은 전세포사멸(proapoptotic) 단백질인 Bax 및 항세포사멸(antiapoptotic) 단백질인 Bcl-2의 조절을 통해 결국 세포 사멸을 억제하는 것으로 나타났다. 시스플라틴 군(CP group)은 Bax 단백질 발현량이 대조군(control group)보다 1.6 배 이상 높았으나, ALA를 처리 세포군에서는 Bax 단백질 발현을 유의하게 완화시켰다(도 7A 및 도 7B). 반대로, 시스플라틴 처리는 Bcl-2 단백질 발현을 하향 조정하였고 ALA 처리 세포는 Bcl-2 단백질 발현을 회복시켰다(도 7C 및 도 7D).ROS scavenging of ALA has been shown to eventually inhibit apoptosis through the regulation of Bax, a proapoptotic protein, and Bcl-2, an antiapoptotic protein. In the cisplatin group (CP group), the Bax protein expression level was 1.6 times higher than that of the control group, but the Bax protein expression was significantly attenuated in the ALA-treated cell group (FIGS. 7A and 7B). Conversely, cisplatin treatment down-regulated Bcl-2 protein expression and ALA-treated cells restored Bcl-2 protein expression (FIGS. 7C and 7D ).
여기서 중요한 것은 비록 세포가 시스플라틴에 의해 이미 손상되었지만(ALA 후처리 군(ALA post-treatment group)의 경우), ALA는 Bax 단백질 발현을 성공적으로 억제하여 세포사멸(apoptosis)을 줄인다는 사실이다. 즉, 이는 ALA가 시스플라틴에 의한 이독성 난청 발병 후에 치료제로서 효과가 있다는 것을 의미한다.What is important here is that although the cells have already been damaged by cisplatin (in the case of the ALA post-treatment group), ALA successfully inhibits Bax protein expression, reducing apoptosis. That is, this means that ALA is effective as a therapeutic agent after the onset of ototoxic hearing loss caused by cisplatin.
<실시예 5> 글루타티온의 산화-환원 반응에 관여하는 ALA 확인<Example 5> Confirmation of ALA involved in the oxidation-reduction reaction of glutathione
대표적인 산화 방지제인 글루타티온(glutathione)의 산화 및 환원에 ALA가 관여하는 것으로 알려져 있다. 글루타티온의 환원된 형태인 GSH는 글루타티온 퍼옥시다아제(GPx)에 의해 글루타티온 디설파이드(Glutathione disulfide; GSSG)로 산화되어 세포 내 ROS를 감소시키며, GSSG는 글루타티온 환원 효소(glutathione reductase; GR)에 의해 GSH를 감소시킨다. 글루타티온 레독스 사이클(glutathione redox cycle)은 GSSG의 산화 형태를 GSH로 감소시키는데 핵심적인 역할을 하는 것으로 알려져 있으므로 웨스턴 블롯에 의한 GPx 및 GR의 발현 수준을 확인하였다.It is known that ALA is involved in the oxidation and reduction of glutathione, a representative antioxidant. GSH, a reduced form of glutathione, is oxidized to glutathione disulfide (GSSG) by glutathione peroxidase (GPx) to reduce intracellular ROS, and GSSG reduces GSH by glutathione reductase (GR). Let it. Since the glutathione redox cycle is known to play a key role in reducing the oxidized form of GSSG to GSH, the expression levels of GPx and GR were confirmed by Western blot.
놀랍게도 GPx 발현이 ALA를 처리한 모든 군에서 4-5 배 정도 증가하였으며, 이는 ALA에 의해 유도된 GPx 발현은 ROS를 능동적으로 제거할 수 있음을 시사한다(도 8A 및 도 8B). 흥미롭게도, GR 발현의 증가는 GPx 발현에 비해 유의하지 않았다(도 8C 및 도 8D). 이것은 GR의 불충분한 발현으로 인해 증가된 GSSG를 GSH로 감소시킬 수 없음을 나타낸다. Surprisingly, GPx expression increased by 4-5 times in all groups treated with ALA, suggesting that GPx expression induced by ALA can actively eliminate ROS (Figs. 8A and 8B). Interestingly, the increase in GR expression was not significant compared to GPx expression (FIGS. 8C and 8D ). This indicates that increased GSSG cannot be reduced to GSH due to insufficient expression of GR.
그러나, GR 발현이 유의하게 증가하지는 않았지만, ALA 전처리 군(ALA pre-treatment group) 및 ALA 후처리 군(ALA post-treatment group)의 GSSG/GSH 비율은 대조군(control group)과 유사하였다(도 8E). 이러한 결과는 ALA가 GSSG를 GSH로 감소시키기 위해 GR의 역할을 수행하여 ROS를 지속적으로 줄이는 역할을 한다는 것을 의미한다.However, although GR expression did not increase significantly, the GSSG/GSH ratio of the ALA pre-treatment group and ALA post-treatment group was similar to that of the control group (Fig. 8E). ). These results imply that ALA plays a role of GR to reduce GSSG to GSH, thereby continuously reducing ROS.
상기 결과들로부터 알파-리포익산(alpha-lipoic acid)은 시스플라틴에 의한이독성 난청을 개선시키는데 효과가 있음을 확인할 수 있었다. 따라서, 본 발명에 따른 알파-리포익산(alpha-lipoic acid)은 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물 또는 개선용 건강기능식품으로 사용될 수 있다.From the above results, it was confirmed that alpha-lipoic acid is effective in improving ototoxic hearing loss caused by cisplatin. Therefore, alpha-lipoic acid according to the present invention can be used as a pharmaceutical composition for treating ototoxic hearing loss caused by cisplatin or as a health functional food for improvement.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.
Claims (5)
A pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin containing alpha-lipoic acid as an active ingredient.
상기 조성물은 청각유모세포 사멸을 억제시키는 것을 특징으로 하는 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물.
The method of claim 1,
The composition is a pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin, characterized in that inhibiting the death of auditory hair cells.
상기 조성물은 청각유모세포에서 Bax 유전자 또는 단백질 발현은 감소시키고, Bcl-2 유전자 또는 단백질 발현은 증가시키는 것을 특징으로 하는 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물.
The method of claim 1,
The composition is a pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin, characterized in that the Bax gene or protein expression is reduced and the Bcl-2 gene or protein expression is increased in auditory hair cells.
상기 조성물은 청각유모세포에서 GPx 유전자 또는 단백질 발현을 증가시키는 것을 특징으로 하는 시스플라틴에 의한 이독성 난청 치료용 약학적 조성물.
The method of claim 1,
The composition is a pharmaceutical composition for the treatment of ototoxic hearing loss by cisplatin, characterized in that increasing the expression of the GPx gene or protein in the auditory hair cells.
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