말뼈추출물은 다양한 골질환의 예방과 치료에 탁월한 효능이 있다고 이전에 보고되었다. 하지만 말뼈 추출물의 다른 약리학적 효능에 대해서는 아직 자세히 밝혀지지 않고있다. 본 연구에서는 말뼈추출물이 중요한 항산화 인자인 hemeoxygenase-1(HO-1)의 발현을 상승시킬 수 있는지, 만약 발현이 증가한다 면 HO-1의 상향 조절이 대식세포에서 항염증 효과를 매개할 수 있는지에 관하여 조사하였다. 이를 위 해서 nitric oxide(NO) 농도측정, 세포 생존능 측정, DPPH 라디칼 소거능 검사를 시행하였다. 또한 염 증성 사이토카인 유전자 발현과 단백질 발현을 측정하기 위해 real time PCR과 Western blotting을 시행하였다. 말뼈추출물은 lipopolysaccharide(LPS, 0.1μg/ml)로 자극한 대식세포주인 RAW264.7 세포 에서 어떠한 세포독성 없이 NO의 생성을 유의성 있게 억제하였으며 inducible nitric oxide(iNOS)와 cyclooxygenase 2(COX-2)의 발현을 억제하였다. 뿐만 아니라 말뼈 추출물은 염증성 사이토카인인 tumor necrosis factor(TNF)-α와 interleukin(IL)-1β의 발현을 억제하였으며 ERK, JNK 및 p-38 MAPK의 단백질 인산화를 억제하였다. 그리고 말뼈추출물은 HO-1과 NF-E2-related factor-2 (Nrf-2) 의 발현을 증가시켰고 이것은 말뼈추출물이 가지고 있는 항 염증효과를 매개할 수 있는 것으 로 보인다. 즉, 말뼈추출물이 HO-1의 발현을 상향 조절한 반면 ERK1/2의 신호전달 경로에 손상을 주 는 것으로 확인되었으며 이러한 말뼈추출물의 효과가 최종적으로 세포손상과 세포의 과산화 자극으로부 터 세포를 보호 할 수 있는 것으로 사료된다.

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level

본 연구는 중간대뇌동맥을 폐쇄한 대뇌허혈성 손상모델에서 ferulic acid에 의해 조절되는 HO-1과 HO-2의 발현에 관하여 조사하였다. 흰쥐(Sprague-Dawley, 수컷)에 ferulic acid (100 mg/kg) 또는 vehicle을 중간대뇌동맥폐쇄술(MCAO) 후 정맥으로 주사하였고 중간대뇌동맥폐쇄술(MCAO)을 실시한 24시간 후 대뇌피질의 조직을 적출하였다. Hematoxylin과 eosin 염색을 통하여 MCAO로 유도된 뇌 손상시 ferulic acid의 보호효과를 확인하였다. MCAO을 시행한 대뇌피질에서는 응축된 핵과 신경세포의 괴사 소견을 보였으나, ferulic acid 투여군에서는 이들 신경세포의 병변을 현저히 완화시켰다. HO-1과 HO-2의 RNA와 단백질 발현의 변화를 reverse-transcription PCR과 Western blot으로 분석하였다. HO-1 발현은 MCAO 후 vehicle 투여군에서 현저히 감소하였으나, MCAO 후 ferulic acid를 투여한 실험군에서는 이들 감소의 완화를 보였으며, MCAO를 시행하지 않은 실험군의 수준으로 유지되었다. 그러나, HO-2의 발현은 MCAO 후 vehicle 투여군과 ferulic acid 투여군에서 유의적인 차이는 관찰되지 않았고 MCAO를 시행하지 않은 실험군의 수준으로 유지되었다. 따라서, 본 연구의 결과는 허혈성 뇌 손상시 ferulic acid는 HO-1 발현을 조절하였으나, HO-2의 발현에는 영향을 미치지 못함을 확인하였다. 결론적으로, 허혈성 뇌손상시 ferulic acid는 HO-1의 발현을 조절하여 신경세포를 보호하는 역할을 수행한다는 사실을 확인하였다.

The present study aimed to verify the effects of DFO on PDL cells, with particular emphasis on focusing on osteoblastic differentiation. Its mechanisms related to heme oxygenase-1 (HO-1) pathway were also analyzed. DFO increased the expression of HO-1 and early osteoblastic differentiation markers, such as alkaline phosphatase (ALP) and bone sialoprotein (BSP). DFO upregulated heme oxygenase-1. Treatment with HO-1 siRNA blocked the DFO-stimulated osteoblastic differentiation and HO-1 expression. The NF-kB inhibitor pyrrolidine dithiocarbamate, phosphatidylinositol 3-kinase inhibitor Wortmannin, and p38 MAPK inhibitor U0126 blocked the effects of DFO on HO-1 expression and osteoblastic differentiation in PDL cells. Collectively, these data suggest that DFO promotes osteoblastic differentiation and induces the expression of defense protein HO-1 probably via PI3K, p38 MAPK, and NF-kB signalling pathways in PDL cells.

Substance P (SP) is known to be expressed in the nerve fibers of dental pulp and periodontal tissues. It was recently reported that SP expression increased in response to orthodontic force. In the present study, we investigated the effect of SP on expression of mineralization markers and heme oxygenase-1 (HO-1) in human immortalized periodontal ligament (IPDL) cells. Cell viability was measured using a 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of mineralization markers, including alkaline phosphatase (ALP), osteonectin (ON) and bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. SP did not significantly change human IPDL cell viability, with the exception of the 24 hour treatment group. Treatment of human IPDL cells with 10-10 to 10-⁴M SP upregulated mineralization marker and HO-1 expression in a time- and concentration-dependent manner. Our results suggest that SP may modulate osteoblastic cell differentiation of human IPDL cells through a mechanism involving HO-1 expression.

Heme oxygenase-l (HO-l) exhibits cyt oprotective effects in many different cell types and is induced by nicotine exposure in human gingival fibroblasts‘ However‘ therole of HO- l in cancer cells exposed to nicotine has not previously been descnbed We investigated the effects of nicotine on HO-l protein expression and cell viability in immortalized (IHOK) and malignant (HN12) human ora l keratinocyte cells using the MTT assay and Western blotting. We al so examined the involvement of t he phosphoinosit ide-3-0H- kinase (PI3K), mitogen-acti vated protein kinase (MAPK) , and nucJear factor-κ B (NF-κ B) signaling pathways in nicotine-induced cytotoxicity and HO- l levels in IHOK and HN12 cell s‘ Nicotine induced HO- l pro ducti on and had cytotoxic effects on cells in both a concentration- and time-dependent manner. Nicotine-induced cytotox icity and accumulation of HO- l were greater in JJ-IOK cells than in HN12 cells Molecular inhibitors of the ERK, p38 MAP kinase, PI3K, and NF-κ B signaling pathways blocked the cytotoxic effects and induction of J-IO-l expression by nicotine. Treatmen t with an t ioxida nts (bil irubin, N-acetyl cysteine) protected cells against nicotine-induced cytotoxicity and blocked the upregula tion of J-IO- l, the effects of which were more pronounced in II-IOK cells than in HN12 cells Collecti vely, these results suggest that J-IO- l plays a principal role in the protective response to nicotine in oral cancel and immortalized keratinocytes. Moreover, the addition of exogenous antioxidants may help to protect oral epithelial cells as chemopreventive agents against nicotine-induced oxidative stress.

Heme oxygenase-l (HO-l) exhibits cyt oprotective effects in many different cell types and is induced by nicotine exposure in human gingival fibroblasts‘ However‘ therole of HO- l in cancer cells exposed to nicotine has not previously been descnbed We investigated the effects of nicotine on HO-l protein expression and cell viability in immortalized (IHOK) and malignant (HN12) human ora l keratinocyte cells using the MTT assay and Western blotting. We al so examined the involvement of t he phosphoinosit ide-3-0H- kinase (PI3K), mitogen-acti vated protein kinase (MAPK) , and nucJear factor-κ B (NF-κ B) signaling pathways in nicotine-induced cytotoxicity and HO- l levels in IHOK and HN12 cell s‘ Nicotine induced HO- l pro ducti on and had cytotoxic effects on cells in both a concentration- and time-dependent manner. Nicotine-induced cytotox icity and accumulation of HO- l were greater in JJ-IOK cells than in HN12 cells Molecular inhibitors of the ERK, p38 MAP kinase, PI3K, and NF-κ B signaling pathways blocked the cytotoxic effects and induction of J-IO-l expression by nicotine. Treatmen t with an t ioxida nts (bil irubin, N-acetyl cysteine) protected cells against nicotine-induced cytotoxicity and blocked the upregula tion of J-IO- l, the effects of which were more pronounced in II-IOK cells than in HN12 cells Collecti vely, these results suggest that J-IO- l plays a principal role in the protective response to nicotine in oral cancel and immortalized keratinocytes. Moreover, the addition of exogenous antioxidants may help to protect oral epithelial cells as chemopreventive agents against nicotine-induced oxidative stress.

Previous in vi tro studies demonstrated that H202 or carbamide peroxide cou ld penetrate i nto pul p chambers through enamel and dentin (Benetti et a l., 2004; G okay et a l. , 2004‘ Suli eman et al .. 2005) ‘ Recently. Lee et al.(2006) demonstrated that H20Z enhanced the diffe rentiation of odontoblast like cell line, whereas it inhibited osteogenic diffe rentiation in pre 。steobl astic cell line, as seen by its efl"ecLs on an early difï"erentiation marker. ALP activity. I-lowever. the effects of HZ02 have not been well elucidated in primary cultured human pulp cells ln th is study‘ we investigated whether HO- 1 is involved in H20 2-induced cytotoxicity and examined the production 0 1" dent in sia lophosphoprotein (DSPP) and other minera li zation markers, in human pulp cells H20Z dec1'eased cell viabili ty. but increased HO-l and DSPP expression in a concentra t ion and time dependent manner. Inhibitors of guanylate cyclase, PI3K. ERK, and p38 MAP kinase blocked J-!?,0 2- induced cytot oxicity and the expression of HO-1 and DSPP mRNAs in pulp cells. These data suggest that t he induction of HO-l by H202 in pu lp cells plays a protective role against the cytotoxic effects of H202 and stimulates DSPP expression. resulting in prematu re oclontoblast differentiation th rough pathways t hat involve cGMP. p38. ancl ERK

The aim of t his study was to investigate the cytotoxic and ni t ric oxide (NO)-inducing effects of bismuth oxide (Bi203)-containi ng Portland cement (BPC) on human dental pulp cells. We also assessed whether heme oxygenase-l (HO-l) is involved in BPC-induced cytotox.icity in dental pulp cells Cytotoxicity and NO production induced by BPC were higher than those induced by Portland cement (PC) at 12 and 24 hours, and the former grad ua lly decreased to the level observed for PC. HO- l and inducible nitric oxide synthase (iNOS) mRNA expressions in the BPC group showed maximal increase at 24 hours. and it gradually decreased with increasing cultivation tlme Hemin treatment reversed the BPC-induced cytotoxicity ‘ whereas zinc protoporphyrin IX treatment increased the cytotoxicity. These results suggested that NO production by BPC correlates with HO-l exp1'ession in dental pulp cells Moreover ‘ BPC- induced HO-l expression in dental pulp cells plays a protective 1'ole against the cytotox.ic effects of BPC.

Although substance P (SP). a potent pro-inflammatory peptide, is involved in inflammation and immune responses, the effect of SP 011 the expression of macl'ophage inJlammatol'Y protein 3a (MIP-3a. CCL20) in periodontal ligament (PDL) cells a l'e unknown Equally as enigmatic is the link between SP. the stress protein heme oxygenase-l (HO-l) , and CCL20 product ion. We investigated whether SP induces the release of chemokine CCL20 from irrunortalized POL (IPDL) cells. and further claif’y SP mediated pathways . We also exarnined the relationship between HO-l and CCL20 by treating POL cells with SP Incubating IPOL cells with SP incl'eased ex pl'ession of CCL20 mRNA and CCL20 protein in a dose-time dependent manner. Highly selective p38 and ERKl/2 inhibitors abl'ogated SP-induced expression of CCL20 lD IPOL cells SP is also responsible fo l' ini tiating phosphorylation of I/( B‘ degl'adation of IK B. and activation of NF-/( B. SP induced expression of HO-l in both a concentration- and time-dependent manner. and CCL20 refl ected similal' patterns. The inductive effects of SP on HO-l and CCL20 were enhanced by HO- l inducer hemin and the membrane-permea ble cGMP analog 8-bromo-cGMP Conversely, this pathway was inhi bited by the HO-l inhibitor zinc Pl'otoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase‘ 1H- [1. 2. 4]uxad iazole[4, 3-alquinoxal i n- 1-one (ODQ) We report hel'ein the pathway that connects SP a long with other modulators 0 1' neuroimmunoregulationto the induction of HO-1 and the inflanunatol'y mediatol' MIP- 3a /CCL20 in IPDL cel ls. which play an impol'tant role in the development 0 1' pe- I'iodontitis or inflammation during ol'thodontic tooth movement

We have examined the effect of NO donor, S-nitl‘ oso-N-acetyl-DL-penicillamine(SNAP) on heme oxygenase-1 (HQ-l) ex pression in human oral immortalized & malignant keratinocytes, and investigated in the control of keratinocyte proliferation evidence tha t HO-1 cou ld be involved in a low dose of NO, NO inhibitor, HOinducer, and HO inhibitor medi ated cytoprotect ion against cytotoxi city induced by a high dose of NO Oral keratinocyte growth inhibitory or anti-proliferative effects were exerted by with SNAP and hemin in a dose- and cul tivation time dependent manner The level of HQ-1 protein was increased in all cell types after exposure hernin dose, and the hemin induced HQ-1 protein achieved at higher maximum level by 12 hrs in all kind of cells , The pretreatment of cells with 0, 2 μ M SNAP reduced 1 mM SNAP-induced death in IHOK and HN4 cells , These cytoprotective effects on high dose of NO induced HQ-1 expresion and cell ular toxicity were blocked by low dose of SNAP, HCB, and ZnPP IX supporting the involvement of HQ-1 in high dose NO induced growth arrest or cell death, But these cytoprotection pattern is different from immortalized and malignant keratinocytes , These results indirectly demonstrate that HQ-1 could be involved in cytoprotection by NO priming against high dose NO induced cytotoxicity in immortalized and maigla nt oral keratinocytes, Thus, HQ-1 might be an important cellular target of NO donor, with clinical implications for the pre vention of inJlammatory di seases and anti-tumor immunity

Al though the changes in tooth morphology and hardness by hydrogen peroxide(H20 z) have been r‘epor‘.ted .‘ the pαr。o야t뻐ec야tive role of heme oxygenase-l(HO-l) against the cytotoxic effects of H202 has not been clarifïed i n human pulp cells ln this st udy. we investigated whether HO-l is involved in Hz0 2-induced cytoLox icity a nd examined the production 0 1' dentin sia lophosphoprotein(DSPP) and other mineralization markers‘ in hllman pu lp cells H202 decreased cell viabi lity, but increased HO-l and DSPP expression in a concentra tion and time dependent manner . Inhibitors of gllanylate cyclase. PI3K, ERK. and p38 MAP kinase blocked H202-indllced cytotoxicity and the expression of HO-l and DSPP mRNAs in pulp cells. These data suggest that the induction of HO-l by H202 in plllp cells plays a protective role against the cytotoxic effects 0 1' HzOz and stimulates DSPP expression‘ reslllting in prematllre odontoblast dilTerentiation throllgh pathways that involve cGMP‘ p38. and ERK.

Al t hough substance P(SP) , a potent pro- inflammatory peptide, is involved in inflammation and immune responses‘ t he eff'ect of SP on t he expression of macrophage inflammatory protein 3a (MIP- 3α CCL20) in periodontal liga ment(PDL) cell s a re unknown, Equally as enigmatic is the link between SP, t he stress protein heme oxygenase- l(HO-l) ‘ and CCL20 procluction, We investigated whether SP induces the release of chemokine CCL20 from immortal ized PDL(IPDL) ceJJ s‘ and fur ther c l a꺼 SP mediated pathways, We also examined the relationship between HO-l a ncl CCL20 by t reating PDL cells with SP, Incubating IPDL cells with SP increased expression of CCL20 mRNA a nd CCL20 protein in a dose-time dependent manner Highly selective p38 and ERKl/2 inhibitors abrogated SP-induced expression of CCL20 in IPDL cell s, SP is a lso responsible for ini t iating phosphorylation of I/C B, degradation of Iκ B‘ ancl activat ion of NF'-/C B, SP induced expression of HO-l in both a concentration- and time-dependent man nel ‘ and CCL20 refl ected s imilar patterns, The inductive effects o[ SP on HO- l and CCL20 wer e enhanced by HO- j inducer hemin and the membrane-permeable cGMP analog 8-bromo-cGMP, Conversely, this pathway was inJübited by t he 1-10난 inhi bitor zinc protoporphyrin IX(ZnPP IX) and the selective inl뼈itor of guanylate cyc1ase‘ lH-[l , 2, 4Joxad iazole[4‘ 3-aJquinoxal in-l-one (ODQ) , We report herein the pathway that connects SP along with other modulators 。f neuroimmunoregulationto the induction of HO-l and t he inflammatory mediator MIP-3a /CCL20 in IPDL cell s‘ which play an important role in the development 01' periodontitis or inflamrnation during orthodontic tooth movem

본 논문의 연구목적은 Choroidal neovascularization (맥락막 신생혈관) 모텔에서 HO-1 발현제와 억제재의 영향을 알아보고자 한다. 30마리의 Brown Norway rat을 각각 10마리씩 세 그룹， Hemin treated group, SnPP treated group, Contorl group으 로 나누어서 실험을 진행하였다. Hemin treat group은 10μmol/kg hemin(Frontier Scientific Inc. USA) 을 SnPP treat group은 10 μmol/kg SnPP(Frontier Scientific Inc. USA)을 laser 시술 2 일에서 14 일까지 복강내 주사하였고 Control군은 0.5 mß씩 식 염 수를 주사하였다. 14 일 후 안저사진촬영과 형광안저촬영을 실시하였다. SnPP treated group에서 Hemin treated group보다 더 많은 선생혈관이 생성되었다. Hemin treated group에서는 맥락막 신생혈관 형성의 정도가 정상대조군에 비교하여 저하됨을 알 수 있었다. 본 연구의 결과 HO-1 의 inducer 인 Hemin 이 맥락막의 신생혈관 억제에 영향을 미치는 것으로 보여진다.

This study was to taken to demonstrate the effects of exogenous nitric oxide(NO) on hu rnan pu lp cell s ‘ In volvement of cyclic 3’, 5' -monophosphate(cGMP) in p버 paJ protection induced by herne oxygenase-l (J-lO-l) against NO-induced cytotoxicity , By use of Western blotting and cell viabi lity assay, we have examined the cytotoxicity and J-lO-l induction in pulp cells that were treated with NO donor ‘ S-nitroso-N-acetyl-D, L-penici 1 lamine(SNAP) , We have assessed wheathel' HQ--l contributes the cytoprotective effect against the cytotoxicity caused by NO, and inves tigated the l'elationship between HO-l and cGMP in the s ignaling pathway, SNAP decreased cell via bility but in creased HO-l expl'ession in a concentl'ation- and time一dependent manner in hurnan pu lp cells NO-induced cyto toxicity was inhibited in the presence of the hemin(inducer of HO-l) , whel'eas was en hanced in the pl'esence zinc protoporphyrin IX(ZnPP IX, HO-l inhibitor), thus Lhe NO-induced cytoLoxicity was cOl'related with HO- l expression. R‘ etreatment with a rnemhrane-permeable cGMP analog, 8-bromo-cGMP, restored cell death and enhanced the HO-l protein expression induced by SNAP, ln contrast‘ inhibition of guanylate cyclase by lI-l -[1,2,4] ox adiazole[ 4,3 口]quinoxalin-l-one(ODQ) pretreated pulp cells to 1 mM SNAP resulting in marked cytotoxicity , These findings , demonstrating a link between J-lO-l, regulated thl'ough the cGMP system and NO-induced cytotox.icity in huma띠 p버 p ceJls , suggesti ng a protective 1'ole of HO-l in pulp infl ammatory disease