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Chemical inhibitors destabilize HuR binding to the AU-rich element of TNF-α mRNA
Min-Ju Chae,Hye Youn Sung,Eun-Hye Kim,Mira Lee,Hojoong Kwak,Chong Hak Chae,Sunwoo Kim,Woong-Yang Park 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.11
Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3'UTR to stabilize TNF-α mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-α mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC50) below 10 μM. The IC50 of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 μM, respectively, for binding of HuR protein to TNF-α mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-α mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-α mRNA and decreased levels of secreted TNF-α. From these results, we could find inhibitors for the TNF-α mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs. Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3'UTR to stabilize TNF-α mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-α mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC50) below 10 μM. The IC50 of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 μM, respectively, for binding of HuR protein to TNF-α mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-α mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-α mRNA and decreased levels of secreted TNF-α. From these results, we could find inhibitors for the TNF-α mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs.
저산소성 허혈성 뇌손상을 유발시킨 어린 백서에서 Myeloperoxidase 측정 검사
양혜정(Hae Joung Yang),피대훈(Dae Hun Pee),임지혜(Ji Hye Lim),최병민(Byung Min Choi),이기형(Kee Hyoung Lee),은백린(Baik-Lin Eun) 대한소아신경학회 2002 대한소아신경학회지 Vol.10 No.2
목 적 : 예전에는 뇌경색 부위에서 발견되는 백혈구를 단지 뇌손상에 뒤따르는 생리적인 반응으로만 생각하였으나 최근 10년 사이 재관류 손상에서도 백혈구에 의한 염증 작용이 중요한 역할을 한다고 알려지면서 이들의 역할에 대한 연구가 새롭게 이루어지고 있다. PML의 존재는 세포질내 과립에서 분비되는 MPO를 생화학적으로 측정함으로서 증명할 수 있는데 저자들은 미성숙 뇌에서 저산소성 허혈증에 의한 뇌손상이 발생할 때 백혈구가 침윤되는 과정을 MPO 측정 검사를 통하여 관찰하였고, P와 L-selectin 억제제인 Fucoidin을 투여한 후 MPO의 변화를 확인하였다. 방 법 : 생후 7일된 어린 흰쥐를 사용하여 우측총 경동맥을 전기 응고시켜 자르고 8% 산소에 노출시켰다. 저산소 노출 후 회복 시간에 따라 동물을 희생시키고 뇌를 추출하여 뇌조직 1 g당 10mL의 20 mM potassium Phosphate buffer(pH 7.4)를 첨가하여 50초간 분쇄하였다. 각각의 균질회된 조직 샘플들은 4℃에서 20분 동안 원심 분리하여 상층액을 제거한 후, 침전물은 처음 조직량에 따라 조직 1 g당 10 mL의 0.5% cetylditrimethylethyl ammonium bromide(wt/vol)가 첨가된 50 mM potassium phosphate buffer(pH 6.0)를 첨가하였다. Vortex를 이용하여 buffer와 침전물을 잘 섞고 60℃수조에서 120분 동안 방치하였다. 그 후 각 조직 샘플들을 4 watts에서 음파처리하고 4℃에서 15분 동안 원심 분리를 시행하여 상층액을 새로운 tube에 옮겼다. MPO 측정검사를 위한 MPO reaction buffer는 50 mM potassium phosphate buffer(pH 6.0) 100 mL에 0.53 mM odianisidine edihydrochlorde와 0.0005% HO를 섞어 만들었다. MPO reaction solution 2.9 mL에 각각의 MPO sample 0.1 mL을 첨가하여 460 nm의 파장에서 5분 동안 MPO에 다른 흡광도 차이를 관찰하였다. 결 과 : 조직내 백혈구 침윤의 지표로 측정한 MPO 활성도는 대조군에서는 미미하였으나 총 경동백을 절단한 우측 대뇌군에서는 허혈 및 저산소증 유발 후 8시간째부터 현저히 증가되기 시작하여 24시간 경과 후 발현이 가장 높았으며, Fucoidin 50 mg/kg으로 전처치한 약물 실험군에서는 MPO 활성도가 현저히 감소하였다. 결 론 : 백혈구는 미성숙 뇌의 저산소성 허혈증에 의한 뇌손상에서 중요한 매개체 역할을 하며, 백혈구의 생화학적 활성도를 나타내는 MPO 활성도는 저산소성 허혈성 뇌손상의 정도를 측정하는 지표로 이용될 수 있다고 생각된다. Purpose : Neutrophils found around an infarcted area in the brain was once considered as only the physiologic response following the brain injury, but recent studies have shown that inflammatory responses by neutrophils play an important role in the reperfusion injury. The presence of ploymorphonuclear leukocytes(PML) is proven by biochemical assay of myeloperoxidase(MPO) Secreted in the cytoplasmic granules. We observed the process of PML infiltration on hypoxic-ischemic brain injury of immature rats by the assay of MPO activity and changes of the MPO activity after the administration of fucoidin, inhibitor of P- and L-selectin. Methods : We used a well characterized model of the brains of 7 day-old-rats, which had unilateral hypoxic and ischemic injuries(HI). Those injuries were induced by unilateral carotid artery ligation followed by timed exposure to hypoxic inspiratory gas mixture(8% O). MPO activity was measured in the brain tissue homogenates of HI rats(n=18) at 0, 2, 8, 24 and 48 hrs and in rats that received fucoidin immediately before and again after hypoxia(50 mg/kg, n=6) at 8 and 24 hrs. Controls(n=2) were rats with neither hypoxia nor ischemia. The brain samples were homogenized in 20 mM potassium phosphate buffer(pH 7.4) for 50 secs. The homogenate was centrifuged at 14,000 g at 4℃ for 15 mins and the supernatant was discarded. The tissue was pulverized, weighed, and suspended in 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) contatining 0.5% cetylditrimethylammonium bromide(wt/vol). The tissue was sonicated and centrifuged at 10,000 g for 15 mins. 200 µL of the supernatant was mixed with 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) containing 10 μL of 1.325 mM o-dianisidine hydrochloride and 170 µL of 3% hydrogen peroxide(vol/vol). Changes in absorbance at 460 nm were measured for 5 mins by using microplate reader. One unit of MPO activity was defined as that degrading 1 µmol peroxide/min at 25℃, and the result was expressed as units of MPO/100 gm tissue. Results : In HI rats, MPO activity increased at 2 hrs after HI and peaked at 24 hrs in the right hemisphere. In rats with fucoidin treatment immediately before and again after hypoxia, the MPO activity significantly decreased in both hemispheres compared with HI rats(P<0.05). MPO activity in the tissue of control rats was insignificant. Conclusion : The dynamic changes of the MPO activity suggest the important role of PMN on hypoxic-ischemic brain injuries in immature rats. MPO activity could be used as an index of the severity of injuries of hypoxic-ischemic brains.
Anti-inflammatory activity of ethylacetate fraction of <i>Cliona celata</i>
Yang, Ju Hae,Suh, Seok-Jong,Lu, Yue,Li, Xian,Lee, Yeun-Kyung,Chang, Young-Chae,Na, Min Kyun,Choi, Jung-Hye,Kim, Cheorl-Ho,Son, Jong-Keun,Chang, Hyeun Wook Informa Healthcare 2011 Immunopharmacology and immunotoxicology Vol.33 No.2
<P>We evaluated the ability of the ethylacetate fraction of marine sponge, <I>Cliona celata</I> (ECC), harvested from Korean seaside to regulate the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells. ECC dose-dependently inhibited both the expression of iNOS protein and mRNA, resulting in decreased production of nitric oxide (NO), with an IC<SUB>50</SUB> of 80.5 μ關g/mL. To investigate action mechanism by which ECC inhibits NO production and iNOS expression, we examined the activation of Iκ觀B in LPS-stimulated RAW264.7 cells. ECC clearly inhibited translocation of nuclear factor-κ觀B (NF-κ觀B) p65 subunits from cytosol to nucleus, which correlated with its inhibitory effects on Iκ觀B-α慣 phosphorylation and degradation. Furthermore, ECC potently suppressed both the reporter gene expression and DNA-binding activity of NF-κ觀B, which was associated with decreased p65 protein levels in the nucleus. Here, we show for the first time that ECC inhibits NF-κ觀B activation through the inhibition of Iκ觀B degradation.</P>