• Tuberculosis and Respiratory Diseases
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• 제43권6호
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• pp.925-935
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• 1996

연구목적: ARDS 발병기전의 일단을 알아보기 위하여 실험동물에서 ARDS와 같은 병변을 유도하는 N-nitroso-N-methylurethane(NNNMU)로 흰쥐에서 급성 폐손상을 유도하였다. ARDS에서의 제 II형 폐포세포의 기능 및 형태학적인 변화가 폐포강 내로의 단백질 이동과 관계 있다고 생각되므로 surfactant를 정량하고 동시에 gamma glutamyl transpeptidase(GGT)의 활성도를 측정하였다. 그리고 백혈구와 연관된 free radical의 역할을 알아보고자 phospholipase $A_2$ ($PLA_2$)의 활성도도 측정하였다. 이때 $PLA_2$의 inhibitor인 dexamethasone을 이용하여 $PLA_2$가 ARDS 발병에 미치는 영향을 알아보고자하였다. 방법: NNNMU에 의해 나타나는 폐부종의 확인을 위해 체중에 대한 폐장의 무게의 비를 계산하였고, 제 II형 폐포세포의 기능을 알아보기 위하여 surfactant의 양 및 GGT의 활성도를 측정하였다. 또한 염증세포의 침윤 및 제 II형 폐포세포의 형태학적인 변화를 광학 및 전자현미경을 이용하여 확인 하였으며 $PLA_2$의 활성도도 측정 하였다. 또한 이 모든 실험을 dexamethasone을 투여한 군에서도 시행하여 $PLA_2$의 억제로 나타나는 효과를 관찰하였다. 결과: NNNMU로 유도된 ARDS와 유사한 급성폐손상시 dexarnethasone은 폐부종을 감소시키고 GGT 및 $PLA_2$의 활성도를 감소시켰다. 형태학적으로는 염증세포 침윤의 감소가 관찰되었으며 병리학적인 소견도 호전시켰다. 결론: ARDS의 병인 중 $PLA_2$의 활성화가 염증세포의 침윤을 증가시키고 동시에 resprratory burst에 의한 free radical의 생성과 그 작용이 급성 폐손상의 중요한 기전으로 생각된다. 특히 $PLA_2$는 백혈구 세포막의 NADPH oxidase의 활성화에 따른 free radical의 생성뿐만 아니라 lipid medialor의 생성에도 관여하여 폐부종을 야기하는 중요한 요소가 되는데, 과량의 dexamethasone 은 $PLA_2$를 억제함으로써 이러한 변화를 감소시키는 것으로 생각된다.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 추계국제학술대회
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• pp.13-13
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• 2003

Methylmercury (MeHg) is a ubiquitous environmental toxicant that can be exposed to humans by ingestion of contaminated food including fish and bread. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of intracellular $Ca^{2+}$ levels (［$Ca^{2+}$］$_{i}$). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity. MeHg activated the acidic form of sphingomyelinase (A-SMase) and group IV cytosolic phospholipase $A_2$ ($cPLA_2$) downstream of PC-PLC, but these enzymes as well as protein kinase C were not linked to MeHg's toxicity. Furthermore, MeHg produced ROS, which did not cause the toxicity. However, D6O9, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner in MDCK and SH-5YSY cells. Addition of EGTA to culture media resulted in partial decrease of [$Ca^{2+}$］$_{i}$ and partially blocked cell death. In contrast, D609 completely prevented cell death with parallel decreases in diacylglycerol and ［$Ca^{2+}$］$_{i}$. Together, our findings indicated that MeHg-induced toxicity was caused by elevation of ［$Ca^{2+}$]$_{i}$ through activation of PC-PLC. The toxicity was not attributable to the signaling pathways such as $cPLA_2$, A-SMase, and PKC, or to the generation of ROS.

• Archives of Pharmacal Research
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• 제24권6호
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• pp.552-556
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• 2001

Histamine and arachidonic acid (AA) release was measured using the P2-purinoceptor antaongists, phospholipase $A_2{\;}(PLA_2)$ and cyclooxygenase (COX)/lipoxygenase (LOX) inhibitors to determine whether or not ATP-induced histamine release is associated with arachidonic acid (AA) release in rat peritoneal mast cells. ATP increased histamine release in a dose dependent manner, whereas adenosine did not. PPADS (a selective P2X-purinoceptor antagonist) and suramin (a nonselective P2X,2Y-purinoceptor antagonist) inhibited ATP-induced histamine release in a dose dependent manner. However, RB-2 (a P2Y-purinoceptor antagonist) did not block ATP-induced histamine release. Manoalide and oleyloxyethyl phosphorylcholine (OPC), secretory PLA$_2$ inhibitors, also inhibited ATP-induced histamine release dose-dependently. Both COX inhibitors (ibuprofen and indomethacin) and LOX inhibitors (baicalein and caffeic acid) inhibited ATP-induced histamine in a dose dependent manner. ATP significantly increased [$^3H$]AA release by 54%. PPADS and suramin significantly inhibited ATP-induced [3H]Ph release by 81% and 39%, respectively. ATP-induced histamine release was significantly inhibited by a variety of protein kinase inhibitors, such as bisindolmaleimide, genistein, methyl 2,5-dihydroxycinnamate, W-7 and trifluoperazine. Overall, the results suggest that ATP-induced histamine release is in part related to the PLA2-mediated AA metabolism and P2X-purinoceptors.

• Biomolecules & Therapeutics
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• 제20권6호
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• pp.526-531
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• 2012

During our on-going studies to identify bioactive compounds in medicinal herbs, we found that saucerneol F (SF), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis), showed in vitro anti-inflammatory activity. In this study, we examined the effects of SF on the generation of 5-lipoxygenase (5-LO) dependent leukotriene $C_4$ ($LTC_4$), cyclooxygenase-2 (COX-2) dependent prostaglandin $D_2$ ($PGD_2$), and on phospholipase $C{\gamma}1$ ($PLC{\gamma}1$)-mediated degranulation in SCF-induced mouse bone marrow-derived mast cells (BMMCs). SF inhibited eicosanoid ($PGD_2$ and $LTC_4$) generation and degranulation dose-dependently. To identify the molecular mechanisms underlying the inhibition of eicosanoid generation and degranulation by SF, we examined the effects of SF on the phosphorylation of $PLC{\gamma}1$, intracellular $Ca^{2+}$ influx, the translocation of cytosolic phospholipase $A_2$ ($cPLA_2$) and 5-LO, and on the phosphorylation of MAP kinases (MAPKs). SF was found to reduce intracellular $Ca^{2+}$ influx by inhibiting $PLC{\gamma}1$ phosphorylation and suppressing the nuclear translocations of $cPLA_2$ and 5-LO via the phosphorylations of MAPKs, including extracellular signal-regulated protein kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Taken together, these results suggest that SF may be useful for regulating mast cell-mediated inflammatory responses by inhibiting degranulation and eicosanoid generation.

• 한국식품위생안전성학회지
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• 제8권3호
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• pp.157-161
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• 1993

본 연구는 ethanol의 reactive metabolite인 acetaldehyde의 일차 배양혈관 평활근 세포에 대한 독성 발현 양식을 규명하기 위한 연구의 일환으로, prostaglandin 합성과 세포독성과의 관련성 여부를 확인하기 위하여 수행되었다. Acetaldehyde는, 일차 배양 혈관 평활근 세포에서의 prostaglandin 합성을 현저히 증가 시켰으며, 이때, cyclooxygenase activity 는 큰 변화없거나 오히려 감소시키는 경향을 보였다. Cyclooxygenase inhibitor 인 indometancin은 acetaldehyde에 의한 LDH release를 현저히 차단시켰으며, aspirin 및 salicylic acid는 전혀 영향을 주지 못했다. Phospholipase $A_2\;(PLA_2)$ inhibitor로 알려져 있는 dexamethasone은 유의적인 세포 독성 경감 작용을 보이지 못하였으며, Lipoxygenase inhibitor 들인 NDGA, propyl gallate 등은 현저한 독성 경감 효과를 보였다. 이상의 결과로부터 acetaldehyde에 의한 일차 배양 혈과 평활근 세포에서의 prostaglandin 합성 증가는 Cell death에 대한 직접적인 원인이 아님을 추론할 수 있었으며, $PLA_2/lipoxygenase$ inhibitor들의 강력한 세포독성 경감 작용으로 미루어 볼 때, acetaldehyde 에 의한 세포독성은 lipoxygenase 대사 산물 혹은 $PLA_2$의 직접 작용에 기인할 가능성을 확인할 수 있었다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.79-79
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• 1997

자연계에 존재하는 수은중 유기수은은 생태계 먹이사슬을 통하여 체내의 여러장기에 축적되어 조직손상을 일으키는 것으로 잘 알려져 있다. 그러나 이러한 세포독성에 대한 정확한 생화학적 기전에 대해서는 자세히 알려진 바가 없다. 포스포리파아제 $A_2$(PLA$_2$)는 세포막의 인지질로부터 Arachidonic acid (AA)와 Lysophospholipid를 유리시키는 효소로 최근 세포손상과 관련하여 그 역할이 주목되고 있으며, 극히 최근, 일차배양 소뇌신경세포를 이용한 연구에서 메칠수은처리에 의해 세포독성의 지표인 Lactate dehydrogenase (LDH)의 유리와 함께 AA 유리가 증가되는 것이 관찰되었으나 여러형태의 PLA$_2$중 어느형태의 효소가 관련되어 있는지, 또한, 그 자세한 기전에 대해서는 불분명한 점이 많다. 본 연구에서는 신장세포의 일종인 MDCK세포를 이용하여 메칠수은의 처리에 의한 PLA$_2$의 활성화 및 그 생화학적인 기전을 구명하고자 하였다. [$^3$H]AA를 MDCK세포의 배양액에 첨가하여 라벨링한 후 메칠수은을 처리하였을때 [$^3$H]AA가 대조군에 비해 농도의존적 및 경시적으로 현저하게 증가하였으며 동시에 LDH의 유리도 함께 관찰되었다. 이러한 [$^3$H]AA의 유리 증가는 세포질 PLA$_2$에 특이적인 저해제로 알려진 AACOCF$_3$의 전처리에 의해 거의 완전히 억제되었으나 LDH의 유리는 오히려 증가하였다. 또한, 글루타치온(GSH)의 전구체인 NAC (N-Acetyl Cysteine)에 의해 [$^3$H]AA의 유리는 부분적으로 감소하였으나, LDH의 유리는 변함이 없었다. 돼지비장이나 MDCK 세포에서 얻어진 세포질 PLA$_2$에 메칠수은을 직접 처리하였을때는 오히려 PLA$_2$의 활성은 감소되었다. 위의 결과들로부터 메칠수은에 의한 [$^3$H]AA의 유리 증가는 세포질 PLA$_2$효소에 대한 직접적인 작용이 아니라 세포내 -SH기의 차단이나 Oxidative Stress에 의해 간접적으로 활성화되는 것으로 예상되며, 세포질 PLA$_2$에 의해 유리된 AA의 세포독설과 관련된 세포내의 역할에 대해 의문이 제기되었다.

• BMB Reports
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• 제32권1호
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• pp.33-38
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• 1999

The effects of eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acids (DHA, 22:6n-3) on the phospholipase $A_2$ ($PLA_2$)-mediated release of arachidonic acid (AA, 20:4n-6) were studied in kidney microsomes from rats fed diets containing sunflower oil (SO) or fish oil (FO) concentrate for 11 months. The amounts of AA released by the endogenous $PLA_2$ enzyme were significantly lower by 38% in the FO, compared to the SO-fed rats (23.2 nmol versus 60.7 nmol AA released/mg protein/h in the FO- and SO-treated groups, respectively). The FO-derived microsomes released less linoleic acid (LA, 18:2n-6) and adrenic acid (22:4n-6), but larger amounts of the n-3 fatty acids, including EPA, DHA, docosapentaenoic acid (DPA, 22:5n-3), and 20:4n-3 than the SO-derived microsomes. A similar replacement of the AA and adrenic acid with the n-3 fatty acids including EPA and DHA was also observed in the microsomal phospholipid fraction from the FO-fed rats relative to the SO-treated group. The results suggest that the $PLA_2$-mediated release of AA is reduced and that of EPA is increased in compensation for AA decline in kidney microsomes from FO-fed rats (0.7 nmol EPA/mg protein/h versus 22.7 nmol EPA/mg protein/h for the SO and FO-treated groups). Replacement of the n-6 with n-3 fatty acids may explain the reduced synthesis of the AA-derived prostaglandins and the concomitant rise in the EPA-derived prostaglandins observed in kidneys of FO-treated rats.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.57-57
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• 1995

1) 효소원의 조제 : 류마티스 관절염환자의 관절액 및 rat platelet PLA$_2$는 장등의 방법으로 조재하여 사용하였으며, 기질은 1-pal- (1-$^{14}$ C) linoleoyl PE로 하여 Dole 변법으로 효소활성을 측정하였다. 2) Histamine 유리반응 ; Rat복강으로부터 정제한 비만세포를 항원-항체 복합체로 자극하거나, $A_{23187}$등의 처리로 유리되는 histamine 을 methylation 시킨 후 유기용매법으로 추출한 후 scintillation counter로 측정하였다. 결과 : \circled1 천연물로부터 분리한 5종의 biflavonoid (amentoflavone 및 그 유도체)의 PLA$_2$ 저해 활성을 검토한 결과 거의 유사한 IC50 (약 3$\mu\textrm{m}$)을 나타내었다. \circled2 이들 중 amentoflavone은 염증성 PLA$_2$(Group II형 PLA$_2$)에 비교적 특이성을 나타내었다. 또한 제해양식은 비경쟁적 이면서 비가역적이였다. \circled3 비만세포에서 histamine 유리 억제반응은 자극제의 종류에 관계없이 억제작용을 나타내었으며, 기존에 임상적으로 사용되고 있는 Tranilast나 DSCG(disodium chromoglycate)에 비하여 10배 이상 강한 histamine 유리 억제작용을 나타내었다.다.

• The Korean Journal of Physiology and Pharmacology
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• 제4권3호
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• pp.219-226
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• 2000

In order to elucidate the role of platelet activating factor (PAF) in the acute lung injury induced by endotoxin (ETX), activities of phospholipase A2, lyso PAF acetyltransferase and oxidative stress by neutrophilic respiratory burst were probed in the present study. To induce acute lung injury, $100\;{\mu}g$ of E.coli ETX (type 0127; B8) was instilled directly into the tracheae of Sprague-Dawley rats. Five hours after the ETX instillation, induction of acute lung injury was confirmed by lung leak index and protein contents in the bronchoalveolar lavage (BAL) fluid. At the same time, lung phospholipase A2 (PLA2) activity and expression of group I and II secretory type PLA2 were examined. In these acutely injured rats, ketotifen fumarate, known as lyso PAF acetyltransferase inhibitor and mepacrine were administered to examine the role of PAF in the pathogenesis of the acute lung injury. To know the effect of the ETX in the synthesis of the PAF in the lungs, lyso PAF acetyltransferase activity and PAF content in the lungs were measured after treatments of ETX, ketotifen fumarate and mepacrine. In addition, the role of neutrophils causing the oxidative stress after ETX was examined by measuring lung myeloperoxidase (MPO) and enumerating neutrophils in the BAL fluid. To confirm the oxidative stress in the lungs, pulmonary contents of malondialdehyde (MDA) were measured. After instillation of the ETX in the lungs, lung leak index increased dramatically (p＜0.001), whereas mepacrine and ketotifen decreased the lung leak index significantly (p＜0.001). Lung PLA2 activity also increased (p＜0.001) after ETX treatment compared with control, which was reversed by mepacrine and ketotifen (p＜0.001). In the examination of expression of group I and II secretory PLA2, mRNA synthesis of the group II PLA2 was enhanced by ETX treatment, whereas ketotifen and WEB 2086, the PAF receptor antagonist, decreased the expression. The activity of the lysoPAF acetyltransferase increased (p＜0.001) after treatment of ETX, which implies the increased synthesis of PAF by the remodelling of lysoPAF in the lungs. Consequently, the contents of the PAF in the lungs were increased by ETX compared with control (p＜0.001), while mepacrine (p＜0.001) and ketotifen (p＜0.01) decreased the synthesis of the PAF in the lungs of ETX treated rats. The infiltration of the neutrophils was confirmed by measuring and enumerating lung MPO and the neutrophils in the BAL fluid respectively. Compared with control, ETX increased lung MPO and number of neutrophils in BAL significantly (p＜0.001) whereas mepacrine and ketotifen decrerased number of neutrophils (p＜0.001) and MPO (p＜0.05, p＜0.001, respectively). The lung MDA contents were also increased (p＜0.001) by ETX treatment, but treatment with mepacrine (p＜0.001) and ketotifen (p＜0.01) decreased the lung MDA contents. Collectively, we conclude that ETX increases PLA2 activity, and that the subsequently increased production of PAF was ensued by the remodelling of the lyso PAF resulting in tissue injury by means of oxidative stress in the lungs.

• Tuberculosis and Respiratory Diseases
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• 제71권2호
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• pp.97-105
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• 2011

Background: It was hypothesized that the immunomodulating effects of moxifloxacin contribute to ameliorate oleic acid (OA)-induced acute lung injury (ALI) by suppression of cytosolic phospholipase A2 (cPLA2). This was based on observations from experiments on rats associated with neutrophilic respiratory burst, cPLA2 activity, and expressions of cPLA2, $TNF{\alpha}$, and COX-II in the lung. Methods: ALI was induced by intravenous injection of OA in male Sprague-Dawley rats. Five hours after OA injection, protein content in bronchoalveolar lavage (BAL), lung myeloperoxidase (MPO) activity, and numbers of BAL neutrophils were measured. As an index of oxidative stress-induced lung injury, the content of malondialdehyde (MDA) in lung tissues was also determined. Lung histology, immunohistochemistry and determination of activity of cPLA2 in lung tissues were carried out. In addition, Western blotting of $TNF{\alpha}$ and COX-II in lung tissues was performed. Results: The accumulation of neutrophils in the lungs was observed after OA injection. BAL protein was increased along with neutrophilic infiltration and migration by OA. Moxifloxacin decreased all of these parameters of ALI and ameliorated ALI histologically. The increased malondialdehyde (MDA) in the lung by OA was also decreased by moxifloxacin. Moxifloxacin not only suppressed cPLA2 expression in the lungs and neutrophils but also decreased cPLA2 activity in lung tissues of rats given OA. The enhanced expressions of $TNF{\alpha}$ and COX-2 in the lung tissues of rats given OA were also suppressed by moxifloxacin. Conclusion: Moxifloxacin inhibited cPLA2 and down-regulated $TNF{\alpha}$ and COX-2 in the lungs of rats given OA, which resulted in the attenuation of inflammatory lung injury.