백서 패혈증 모델에서 HSP70의 과도 발현이 iNOS의 발현에 미치는 효과에 관한 연구

이용근,안융,임대호,백진아,고승오,신효근,Lee, Yong-Keun,Ahn, Yung,Leem, Dae-Ho,Baek, Jin-A,Ko, Seung-O,Shin, Hyo-Keun 대한구강악안면외과학회 2010 대한구강악안면외과학회지 Vol.36 No.5

Introduction: Heat shock protein70 (HSP70) is a highly conserved family of proteins produced after a variety of stresses. Many studies reported that the overexpression of HSP70 can improve the prognosis of the patients with sepsis through a reduction of the nitric oxide concentration. However, these results only revealed the effect of HSP70 and nitric oxide. No studies have examined the relationship between HSP70 and nitric oxide. The aim of this study was to evaluate the effect of the overexpression of HSP70 on the expression of inducible nitric oxide synthase and the nitric oxide concentration. In addition, the mechanism of the relationship of HSP70 and inducible nitric oxide synthase (iNOS) in sepsis was examined. Materials and Methods: The experiments were performed on male sprague-dawley rats. Sepsis was induced by a cecal ligation and puncture (CLP). Glutamine (GLN) or saline was administered 1 hour after the initiation of sepsis. Serum and lung tissues were acquired from the rats 12 hours or 24 hours after the initiation of sepsis. The nitric oxide concentration, the expression of HSP70 in lung, and the gene expression of iNOS in lung were analyzed. The three groups, sham operation, CLP and CLP+GLN, were compared. Results: Compared to the other groups, in CLP+GLN, GLN administered after the initiation of sepsis enhanced the expression of HSP70 in the lung at 12 hours ($47.19{\pm}10.04$ vs. $33.22{\pm}8.28$, P=0.025) and 24 hours ($47.06{\pm}10.60$ vs. $31.90{\pm}4.83$, P=0.004). In CLP+GLN, GLN attenuated the expression of iNOS messenger RNA (mRNA) in the lung at 12 hours ($5,513.73{\pm}1,051.60$ vs. $4,167.17{\pm}951.59$, P=0.025) and 24 hours ($18,740.27{\pm}8,241.20$ vs. $9,437.65{\pm}2,521.07$, P=0.016), and reduced the concentration of nitric oxide in the serum at 12 hours ($0.86{\pm}0.48$ vs. $3.82{\pm}2.53$, P=0.016) and 24 hours ($0.39{\pm}0.25$ vs. $1.85{\pm}1.70$, P=0.025). Conclusion: The overexpression of HSP70 induced by the administration of GLN in sepsis attenuates the expression of the iNOS gene but reduces the nitric oxide concentration.

패혈증에서 Heat Shock Protein 70의 과도발현이 Inducible Nitric Oxide Synthase의 발현에 미치는 효과

이용근 ( Yong Keun Lee ),신효근 ( Hyo Keun Shin ),권운용 ( Woon Yong Kwon ),서길준 ( Gil Joon Suh ),윤여규 ( Yeo Kyu Youn ) 대한외상학회 2008 大韓外傷學會誌 Vol.21 No.1

Purpose: The aim of this study was to evaluate the effect of overexpression of heat shock protein 70 (HSP70) on the expression of inducible nitric oxide synthase and on the concentration of nitric oxide and to determine the mechanism for the relationship between HSP70 and inducible nitric oxide synthase (iNOS) in sepsis. Methods: Experiments were performed on male Sprague-Dawley rats, and sepsis was induced by using cecal ligation and puncture (CLP). Glutamine (GLN) or saline was administered 1 h after initiation of sepsis. We acquired serum and lung tissues from the rats 12 h or 24 h after initiation of sepsis. We analyzed the concentration of nitric oxide, the expression of HSP70 in the lung, and the gene expression of iNOS in the lung. Results: In CLP+GLN, glutamine given after initiation of sepsis enhanced the expression of HSP70 in the lung at 12 h (CLP+GLN vs. CLP:: 47.19 ± 10.04 vs. 33.22 ± 8.28, p = 0.025) and 24 h (CLP+GLN vs. CLP: 47.06 ± 10.60 vs. 31.90 ± 4.83, p = 0.004). In CLP+GLN, glutamine attenuated the expression of iNOS mRNA in the lung at 12 h (CLP+GLN vs. CLP: 4167.17 ± 951.59 vs. 5513.73 ± 1051.60, p = 0.025) and 24 h (CLP+GLN vs. CLP: 9,437.65 ± 2,521.07 vs. 18,740.27 ± 8,241.20, p = 0.016) and reduced the concentration of nitric oxide in serum at 12 h (CLP+GLN vs. CLP: 0.86 ± 0.48 vs. 3.82 ± 2.53 umol/L, p = 0.016) and 24 h (CLP+GLN vs. CLP: 0.39 ± 0.25 vs. 1.85 ± 1.70 umol/L, p = 0.025). Conclusion: The overexpression of HSP70 induced by the administration of glutamine in sepsis attenuated the gene expression of iNOS and reduced the concentration of nitric oxide.

개소시랑개비 추출물의 RAW264.7대식세포에서 in vitro 항염효과

남정환,김현삼,김병진,유홍섭,장동칠,진용익,유동림,최종근,박희준,이승빈,이경태,박수진,Nam, Jung-Hwan,Kim, Hyun-Sam,Kim, Byoumg-Jin,Yu, Hong-Seob,Chang, Dong-Chil,Jin, Yong-Ik,Yoo, Dong-Lim,Choi, Jong-Keun,Park, Hee-Jhun,Lee, Seung-Bin,Lee 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.1

본 연구에서는 개소시랑개비 (Potentilla supina)의 전초를 이용하여 세포독성 및 항염증 활성 효과를 평가하였다. 대식세포인 RAW264.7 cell에서 염증 매개 물질인 lipopo-lysacchride(LPS)로 염증을 유발해 nitric oxide (NO), Inducible nitric oxide synthase (iNOS)와 prostaglandin$E_2$($PGE_2$) 같은 염증 유발 인자들의 억제효과를 확인하였다. 개소시랑개비 ethyl acetate 분획물의 염증 유발 인자 억제 시 $IC_{80}$ value를 측정하였을 때 nitric oxide 및 prostaglandin $E_2$ 생성을 농도의존적으로 현저하게 저해하는 농도는 각각 29.34와 $50.75{\mu}g/ml$이었고 Inducible nitric oxide synthase의 양도 $50{\mu}g/ml$ 처리하였을때 농도 의존적으로 발현 감소 하였다. 따라서 본 연구결과는 개소시랑개비의 ethyl acetate 분획물과 같은 비극성용매 분획물들이 유의성 있는 항염증 효과를 나타내었으며, 이러한 효능은 예방의학적 가능성을 충분히 가지고 있기에 염증성질환의 예방을 위한 건강 기능성식품의 개발 가능성을 제시할 수 있을 것으로 기대된다. 또한 염증과 관련된 사이토카인 및 단백질 발현 메커니즘에 대한 추가적인 연구가 필요할 것으로 판단된다. Potentilla supina (Rosaceae) has traditionally been used to treat disorders of hemostasis, dysentery, malaria, bloody discharge and arthritis, and it has antinociceptive and anti-inflammatory properties. However, validity of the anti-inflammatory activity has not been scientifically investigated so far. Therefore, the aim of this study was to investigate the anti-inflammatory potential of P. supina using the ethanolic extract of P. supina and its sub-fractions. To evaluate the anti-inflammatory effects of P. supina, we examined the inflammatory mediators such as nitric oxide (NO), inducible nitric oxide synthase (iNOS) and prostaglandin $E_2$ ($PGE_2$) in RAW 264.7 cells. Our results indicated that ethyl acetate fraction significantly inhibited LPS-induced NO, iNOS and $PGE_2$ production in RAW 264.7 cells. This result showed that ethyl acetate fraction of P. supina is expected to be a good candidate for development into a source of anti-inflammatory agents.

지모의 수용성 추출물이 생쥐 소교세포에서 Lipopolysaccharide로 유발된 Cyclooxygenase-2와 Inducible Nitric Oxide Synthase 발현에 미치는 영향

윤종태 ( Jong Tae Yun ),송윤경 ( Yun Kyung Song ),임형호 ( Hyung Ho Lim ) 한방재활의학과학회 2006 한방재활의학과학회지 Vol.16 No.4

목적 : 지모는 임상에서 해열, 항염, 진정, 이뇨 그리고 항당뇨 작용을 가지고 있어 한의학에서 청열자음의 목적으로 사용되어 왔다. 본 연 구는 생쥐 BV2 신경교세포에서 lipopolysaccharide에 의해 유발되는 염증유도산물인 PGs와 NO의 신경염증반응에 대한 지모의 효과를 살펴보기 위해 시행하였다. 방법 : 지모의 항염증 효과를 알아보기 위하여 lipopolysaccharide는 24시간 반응시키는 한편, 지모는 lipopolysaccharide 처치 1시간 전에 전처치 한 이후 reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, PGE2 immunoassay, NO detection 등의 분석방법을 사용하였다. 결과 : lipopolysaccharide는 cyclooxygenase-2 와 inducible nitric oxide synthase의 mRNA와 단백질의 발현을 증가시켜 prostaglandin E2 합성과 nitric oxide 생성을 증가시켰다. 지모를 전처치 하였을 때에는 lipopolysaccharide에 의하여 발현이 증가되던 cyclooxygenase-2와 inducible nitric oxide synthase mRNA와 단백질의 발현이 억제되었고 그 결과 prostaglandin E2 의 합성과 nitric oxide의 생성도 억제되었다. 결론 : 본 연구 결과, 지모는 여러 가지 염증성 질환에 진통 및 항소염 작용이 있는 것으로 보여진다.

Pertussis toxin-induced hyperacute autoimmune encephalomyelitis in Lewis rats is correlated with increased expression of inducible nitric oxide synthase and tumor necrosis factor alpha

Ahn, Meejung,Kang, Jongchul,Lee, Yongduk,Riu, Keyzung,Kim, Yong-sik,Jee, Youngheun,Matsumoto, Yoh,Shin, Taekyun 제주대학교 방사능이용연구소 2001 연구보고 Vol.15 No.-

자기면역성 뇌척수염(experimental autoimmune encephalomyelitis, EAE)은 뇌조직항원을 면역한 후 야기되는 염증성 질병으로 사람 다발성결화증의 한 모델로 연구되고 있다. 자기면역성 뇌염의 시작은 뇌조직항원에 반응하는 림프구가 중추신경계에 침윤되면서 마비를 나타내는데 이 과정 중에는 여러 종류의 pro-inflammatory mediator (tumor necrosis factor-alpha (TNF-α)와 inducible nitric oxide synthase (iNOS)등)가 관여하는 것으로 알려지고 있다. 이 연구에서는 염증의 진행 단계에 따라 염증 유도 또는 염증 억제의 상반된 기능을 갖는 것으로 알려진 TNF-α와 iNOS가 심급성 뇌척수염 진행에 어떠한 영향을 미치는지를 조사하였다. 뇌염을 유도하기 위한 항원으로는 랫트 척수 조직 유제를 complete Freund adjuvant와 혼합하여 뒷 발바닥에 주사하였으며 심한 뇌척수염을 유도하기 위하여 pertussis toxin(500ng/ea)을 면역하는 날 복강내로 주사하고 매일 체중과 마비 정도를 확인하였다. 독소를 주사한 실험군에서는 대조군(11일)에 비해 마비의 시작이 빨랐으며(9일), 대조군은 자연 회복하는 반면 독소룰 주사한 실험군에서는 모두 폐사하였다. 척수 조직 내 TNF-α 와 iNOS의 양적인 변화를 조사하기 위하여 Competitive PCR과 Western blot를 이용하였으며, 세포형을 구분하기 위하여 면역염색을 이용하였다. Competitive PCR결과 TNF-α는 PT를 투여한 자기면역성뇌척수염의 심한 마비기(EAE,G3)에서 PT를 투여하지 않은 대조군보다 약 5배가 증가하였으며(p<0.01), Western blot결과 iNOS는 PT를 투여한 군에서 정상조직에 비해 약 6배가 증가하였고, PT를 투여하지 않은 군에 비해서는 약 3개바 증가하였다(p<0.01). 면역염색결과 PT를 투여하지 않은 랫트보다 투여한 랫트의 척수조직에서 iNOS 양성 세포가 약 15배가 증가하였으며(p<0.01), 또한 연속절편에서 이들 세포가 큰포식세포임을 확인하였다. 이상의 결과를 종합해 볼 때, 자기면역성 뇌척수염의 초기 유도과정에서는 TNF-α와 iNOS는 염증의 약화에 관여됨을 알 수 있었다. The involvement of inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α), which have diverse roles in the progression of autoimmune disease models, was studied in pertussis toxin (PT-induced hyperacute experimental autoimmune encephalomyelitis (EAE) in Lewis rats. The expression of TNF-α mRNA(increased 5 fold, p<0.01) and iNOS protein (3 fold, p<0.01) was much greater in the spinal cords with PT(+) EAE at the peak stage of EAE than in those with PT(-) EAE, as shown by competitive PCR and Western blot analysis, respectively. Immunohistochemistry showed that the majority of EDI-positive macrophages in EAE lesions contained iNOS, and that three were many more iNOS-positive cells in the CNS lesions of PT(+) rats than in those of PT(-) rats. These findings suggest that PT-induced hyperacute EAE is partly mediated by the enhanced expression of iNOS and TNF-α in the early stages of rat EAE.

The Effect of Phorbol Myristate Acetate on the Synthesis of Nitric Oxide in Murine Microglial Cells

Rim, Gyoo-Nam,Kim, Jong-Moon,Chung, Hun-Taeg 대한신경외과학회 1995 Journal of Korean neurosurgical society Vol.24 No.12

본 연구에서는 생쥐 뇌 소교세포에서 phorbol ester가 nitric oxide(NO) 합성에 미치는 효과에 대하여 실험하였다. Protein kinase C(PKC)의 활성자극제로 알려져 있는 phorbol 12-myristate- 13-acetate(PMA)는 생쥐 뇌 소교세포에서 인터페론 감마와 함께 작용하여 NO의 합성을 증가시켰으나 단독으로는 아무론 효과도 나타내지 않았다. 이 세포에서 NO의 합성을 증가시키는 PMA의 가장 효과적인 농도는 PKC를 완전히 활성화시키는 범위의 농도였다. Northern blotting의 결과로 NO합성의 증가는 inducible NO synthase(iNOS) mRNA양의 증가임을 알 수 있었다. PKC 활성억제제인 staurosporine(STSN)이나 polymyxin B등을 세포에 처리하였을 때는 인터페론 감마와 PMA로 자극된 NO합성이 감소 되었다. 게다가 PMA로 전처리된 세포는 PKC활성이 하향 조절되어 인터페론 감마와의 상승작용을 감소시키는 것으로 보아 NO합성에 PKC가 관여하고 있다는 것을 알 수 있었다. L-arginine의 analouge인 N^(G)-monometyl-L-ariginine monohydrate(N^(G)MMA)와 arginase도 인터페론과 PMA에 의해 유도되는 NO생성을 억제시켰다. 본 연구 결롸고 PMA는 iNOS의 발현에 관여하는 것이 아니고 iNOS mRNA의 stability에 관여하여 NO 합성에 상승적인 효과를 나타내는 것으로 보인다. In this study, the effect of phorbol ester on the synthesis of nitric oxide(NO) in murine microglial cells was examined. Phorbol 12-myristate 13-acetate(PMA), a protein kinase C(PKC) activator, alone had no effect, whereas PMA with recombinant interferon-γ(rIFN-γ) synergistically increased NO synthesis in murine microglial cells. The maximal effect of PMA in the increase of NO synthesis always fit with the range for full activation of PKC in these cells. The increase of NO synthesis was reflected as increased amount of inducible NO synthase(iNOS) mRNA by Northern blotting. Treatment of PKC inhibitors such as staurosporine(STSN) or polymyxin B decreased rIFN-γ plus PMA-stimulated NO synthesis. Further, prolonged incubation of the cells with PMA, which down regulate PKC activity, abolished synergistic cooperative effect with IFN-γ. N^(G)-monomethyl-L arginine monohydrate (N^(G)MMA), an analogue of L-arginine, and arginase inhibited rIFN-γ plus PMA-induced NO production in murine microglial cells. On the basis of these observations, we conclude that PKC might not be involved in the expression of iNOS, but instead, might be involved in the post-transcriptional modification of iNOS mRNA.

마우스 종양발생에서 Nitric Oxide의 역할에 관한 연구 Ⅲ : Helicobacter pylori에 의해 유발된 마우스 위암에서 염증매개인자의 역할 The role of inducible nitric oxide synthase and cyclooxygenase-2 in H. Pylori-associated gastric carcinogenesis

남기택,오상연,조현무,이국경,강진석,제정환,최미나,한상욱,김대용,장동덕,양기화,안병우 식품의약품안전청 2001 식품의약품안전청 연보 Vol.5 No.-

feficotorfer fyf☞ri(Hp)가 위암파 관련이 있다는 역학적인 증거는 많이 있지만 이에 대한 정확한 기전에 대해선 밝혀져 있지 않고 있으며 실험동물 모델도 적절하지 못한 것으로 알려져있다. 본 실험에서는 위암의 원인으로 알려져 있는 f, fyforf'(Hp)를 이용하여 마우스에서 위암 모델을 확립하고 만성염증과정 중에 생성되는 리0와 COX-2 등의 발현이 위암발생에 미치는 명향을 통하여 예방과 치료를 위한 점근을 시도하고자 하였다. 마우스를 7군으로 나누어서 1, 2, 3, 4군의 등물은 MNU를 증류수에 200ppm 농도로 음수병득 이용하띤 10주간 격주로 투여하였으며 MHU 음술 투여 1주 휴씩 후 배양한 f. fyrofi 를 약 109cru/rfll 로 맞춰 한 마리당 0.1ml 씩 이틀 간격으로 세 번에 걸쳐 하룻방 금식시킨 1, 2, 3, 5, 6, 7군기 마우스의 위장에 투입하떴다. 균 투입을 마친 후 다응 날부터 2군쏙 6군은 iNOS 억제제인 aminoguanidine(AG)을 음수병으로 툰여하였으며 3군과 7군은 COX-2 척제제인 nimesulide(NSD)를 투여하였다. 위의 종양발생양상을 샅최보면 bfNU와 Hp만을 투여한 1 관 ; (hfNU +Hp), 2군 : iNO을 inhibitor 투여군(MNU+HP+AG'1. 3군 ;CO딘-2 Inilibitor 투여군(MNU누Hp누 NSD), 4군 ,MNlf 단독투여군, 5군 ;Hp 단독투여춘, 6군 ; 러p 단독에 AG투여군, 7군 , Hp 단독에 NSD투여군의 종양발샐을은 각각 쁜.Bff(l1/16), 70.6%f12/ti), 했.9ff(7/18), 10%(1/10), Off(O/IS)0%(O/S), 0%(O/5)의 발쟁율을 보여 iNOS 억제제인 AC은 좁양발생을 억계하지 못하였으며, COX-2억제제인 NSD 는 종양발생을 유의적으로 감소시켰다. 콩양발생개수에서는 2.62±0.36, 1.41츠0.14, 0.44 르0.12, 0.10±0.10을 보여 AC와 NSD에서 유의성 있게 발생개수를 줄였다. Hp 단독투여에 의해즌는 종양발생이 나타나지 않았으며 HP+AG, Hp+ IfSD 추여군에콕 시험증료 시점에 약물에 의해 Hp의 제균효과가 있는지의 여부를 확인끓기 위하여 PCR을 이용하여 확인한 결과 모두 양성인 것으로 나타나 Hp의 제균효과에 의한 촐양발생 억제가 일어나지는 않았다. 위의 결과로 볼 때 Hp는 위암발생을 촉진하는 것으로 나타났고 딘p 감염시 매개되는 염증인자를 억제하였을 때 종양발생을 억제하는 것으로 위암 발생에서 염증매개인자는 종양을 촉진하는 것으로 나타났으며 it,705 억제제쓱 COX-』 억제제의 위알 예밭효과fl는 효과적일 것으로 사료된다 In spite of a large volume of epidemiological evidence indicating significant relationship between H. pylori infection and gastric adenocarcinoma, a doubt still exists on an elevated risk of stomach cancer by H. pylori infection due to lack of direct evidence of their exact mechanistic link. It is, therefore, essential to have an appropriate animal model for detailed analysis of the role of H. pylori played in gastric carcinogenesis. There is a wealth of evidence to support that over production of inducible nitric oxide synthase(iNOS) and cyclooxygenase-2(COX-2) is involved in the pathogensis of various cancer in both rodents and humans. The aim of this study was to establish a mouse model for H. pylori-associated gastric carcinogenesis and to identify the role of inducible nitric oxide synthase(iNOS) and cyclooxygenase-2(COX-2) played during the gastric carcinogenesis in mice. Eighty-three specific pathogen free, six-week-old male C57BL/6 mice were randomly divided into seven groups. Animals of the group 1, 2, 3, 4 were given MNU in their drinking water at the concentration of 200 p.p.m. for total five cycles of one-week regimen with one-week pause. After completion of MNU administration, they were given autoclaved distilled water for one weeks, and groups 1, 2, 3, 5, 6, 7 were inoculated with H. pylori. After completion of H. pylori. inoculation, groups 2 and 6 were given aminoguanidine in their drinking water at concentration of 1000p.p.m. and animals of group 3 and 7 were given the diet containing 200 ppm nimesulide at 12 weeks of age. All animals were killed at 50 weeks of age. The incidences of the glandular stomach tumors in the group 1, 2, 3 and 4 were 87.5%(14/16), 76.4%(13/17), 44.4(8/18), 10.0%(1/10), respectively and the tumor incidence of group 3(MNU→Hp+nimesulide) was significantly lower than those of group 1(MNU→Hp) at the value of P<0.01. The average numbers of tumors of group 2(MNU→Hp+AG : 1.41±0.24) and group 3(MNU→Hp+nimesulide : 0.44±0.12) were significantly lower than those of group 1(MNU→Hp : 2.62±0.36) at the value of P<0.05. Therefore, overproduced iNOS and COX-2 plays an important role in mice gastric carcinogenesis. We concluded iNOS and COX-2 inhibitor have good effects on gastric carcinogenesis.

Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells

Lee, Jin-Sun,Lee, Hong Jae,Lee, Jae Won,Lee, Sang Cheon,Heo, Jung Sun S. Karger AG 2018 CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Vol.51 No.2

<P><B><I>Background/Aims:</I></B> This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). <B><I>Methods:</I></B> We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO<SUB>3</SUB>) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3’,5’ monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. <B><I>Results:</I></B> Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. <B><I>Conclusion:</I></B> These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.</P>

세포주 배양에서 근적외선 조사 배지의 항 염증효과

김상경,신임희,최창혁,최정윤 대한진단검사의학회 2009 Annals of Laboratory Medicine Vol.29 No.4

배경 : 근적외선은 0.8-1.5 μm 파장대의 빛으로 다양한 손상, 감염, 허혈성 질환에 치료로 사용된다고 보고되고 있다. 최근 근 적외선을 물, 섬유 등에 쪼이면 지속적으로 근적외선이 방출되 게 하는 기술이 개발되었다. 본 연구는 근적외선을 적절히 조사 한 배지에서 근적외선이 지속적으로 방출되어 항 염증 효과를 가질 것으로 가정하고, 우선 세포배양에서 근적외선 조사배지가 세포 생존력과 염증성 자극에 대한 반응에 미치는 영향을 알아 보고자 하였다. 방법 : CAPE (혈관내피세포), NIH3T3 (섬유아세포), RD (평 활근세포) 등 세 가지 종류의 세포주를 10% 우태아혈청이 포함 되고, 근적외선이 조사된 배지와 일반배지에서 37℃, 5% CO2 배양기에서 4일간 배양하였다(1×104 cells/well). 근적외선의 항 염증 효과를 보기 위해 배지에 10 mcg/mL의 lipopolysaccharide (LPS)와 sodium nitroprusside (SNP)를 첨가하였다. 세 포증식은 methylthiazol tetrazolium (MTT: 3-[4,5-dimethylthiazole- 2-yl]-2,5-diphenyltetrazolium bromide) assay로 측정하였고, interleukin (IL)-1 beta와 nitric oxide는 ELISA 법, inducible nitric oxide synthase (iNOS)와 cyclooxygenase- 2 (COX-2)는 간접면역형광법을 이용하였다. 결과 : 근적외선 조사배지는 CAPE 세포 증식에 유리하였으 며(N=8, P=0.000), LPS 처리한 NIH3T3 세포에서 IL-1 beta 분비를 감소시켰고(N=4, P=0.026), nitrate 생성을 억제 시키는 경향이 있었으나 통계적으로 유의하지 않았다(N=4, P=0.076). 근적외선 조사배지는 NIH3T3 세포에서 iNOS 발현을 억제하였 으나, COX-2 발현은 두 가지 배지에서 차이가 없었다. 결론 : 근적외선 조사 배지는 일반배지에 비하여 세포배양 시 혈관내피세포의 증식을 촉진시키고, 섬유아세포에는 항 염증 효 과를 나타내었다. 이러한 결과는 임상적으로 염증성 질환에 근 적외선을 적용할 기초자료로 이용될 수 있을 것이다. Background : Near-infrared light (NIR, 0.8-1.5 μm light) has been used in therapeutic devices for various injuries such as infected, ischemic and hypoxic wound. NIR-emitting technology has been developed recently in Korea. We hypothesized that NIR may have an anti-inflammatory effect and investigated the effect of NIR-irradiated media on cell culture. Methods : Three kinds of cell lines, CAPE (vascular endothelial cell), NIH3T3 (fibroblast), and RD (smooth muscle cell) cells were cultured for 4 days in 10% FBS-containing media (1×104 cells/ well), which were irradiated or not irradiated (control) by Eco-NFIR Drive (Model #0210, Ecowavetech, Korea). The cells were stimulated by 10 mcg/mL of bacterial lipopolysaccharide (LPS) or sodium nitroprusside (SNP). Cellular proliferation was measured by methylthiazol tetrazolium assay. Expression of interleukin (IL)-1 beta and nitric oxide was measured by ELISA. Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was measured by immunofluorescence staining. Results : NIR-irradiated medium was favorable for CAPE cell proliferation (N=8, P=0.000). IL-1 beta secretion from LPS-stimulated NIH3T3 cells incubated in the NIR medium was below that of control medium (N=4, P=0.026). Nitrate production seemed to be low in NIR-irradiated medium although statistically insignificant (N=4, P=0.076). Expression of iNOS of the LPS-stimulated cells was decreased in NIR medium, however, Cox-2 expression was not different between the two media. Conclusions : NIR-irradiated medium supported vascular endothelial cell proliferation and showed an anti-inflammatory effect on fibroblast culture. These results can be used as basic data for future research on the clinical application of NIR.

Carrageenin으로 처치한 흰쥐 발 염증에서 Indomethacin에 의한 유도성 nitric oxide synthase의 발현증가

원혜영(Hye Young Won),강건욱(Keon Wook Kang),김영미(Young Mi Pak),김낙두(Nak Doo Kim) 대한약학회 1999 약학회지 Vol.43 No.2

Present study was aimed to examine whether indomethacin affected the production of NO in the rat paw exudate by carrageenin. Paw edema and nitrite/nitrate levels in the paw exudate were maximal after 4h and remained elevated up to 10h, whereas indomethacin (10mg/kg. Po) significantly inhibited the carrageenin-induced paw edema and levels of nitrite/nitrate in the paw exudate. However, paw edema and nitrite/nitrate levels were increased thereafter for 10h. Indomethacin also enhanced the expression of iNOS mRNA and protein 4h after carrageenin injection. Indomethacin inhibited the level of PGE2 in the paw exudate in a time-dependent manner. These results suggest the possibility that indomethacin may potentiate expression of iNOS and subsequently increase nitrite/nitrate level in the late phase of carrageenin-induced rat paw inflammation possibly by suppressing cycloxygenase activity.