Flavocoxid，环氧化酶和 5-脂氧合酶的双重抑制剂，可减弱脂多糖刺激的巨噬细胞中促炎表型的激活。
Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages.
机构信息
Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy.
出版信息
Br J Pharmacol. 2009 Aug;157(8):1410-8. doi: 10.1111/j.1476-5381.2009.00322.x.
BACKGROUND AND PURPOSE
The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.
EXPERIMENTAL APPROACH
LPS-stimulated (1 microg.mL(-1)) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32-128 microg.mL(-1)) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein kappaB-alpha (IkappaB-alpha) levels were evaluated by Western blot analysis. Nuclear factor kappaB (NF-kappaB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-alpha (TNF-alpha) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.
KEY RESULTS
LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 microg.mL(-1)) significantly inhibited COX-2 (LPS = 18 +/- 2.1; flavocoxid = 3.8 +/- 0.9 integrated intensity), 5-LOX (LPS = 20 +/- 3.8; flavocoxid = 3.1 +/- 0.8 integrated intensity) and iNOS expression (LPS = 15 +/- 1.1; flavocoxid = 4.1 +/- 0.4 integrated intensity), but did not modify COX-1 expression. PGE(2) and LTB(4) levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IkappaB-alpha protein (LPS = 1.9 +/- 0.2; flavocoxid = 7.2 +/- 1.6 integrated intensity), blunted increased NF-kappaB binding activity (LPS = 9.2 +/- 2; flavocoxid = 2.4 +/- 0.7 integrated intensity) and the enhanced TNF-alpha mRNA levels (LPS = 8 +/- 0.9; flavocoxid = 1.9 +/- 0.8 n-fold/beta-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.
CONCLUSION AND IMPLICATIONS
Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.
背景与目的
黄芩中的黄酮类化合物黄芩苷和儿茶素分别来自黄芩和金合欢，已被用于各种临床应用。Flavocoxid 是一种混合提取物，含有黄芩苷和儿茶素，作为环氧化酶（COX）和 5-脂氧合酶（LOX）酶的双重抑制剂。本研究通过测量沙门氏菌脂多糖（LPS）刺激的大鼠腹膜巨噬细胞中炎症标志物的蛋白和基因表达，研究了 flavocoxid 对炎症的抑制活性。
实验方法
用不同浓度的 flavocoxid（32-128 μg·mL-1）或 RPMI 培养基与 LPS 刺激的（1 μg·mL-1）大鼠腹膜巨噬细胞共同孵育不同时间。通过 Western blot 分析评估诱导型 COX-2、5-LOX、诱导型一氧化氮合酶（iNOS）和抑制性蛋白 kappaB-α（IkappaB-α）水平。通过电泳迁移率变动分析研究核因子 kappaB（NF-kappaB）结合活性。通过实时聚合酶链反应和酶联免疫吸附试验分别测量肿瘤坏死因子-α（TNF-α）基因和蛋白表达。最后，评估巨噬细胞上清液中的丙二醛（MDA）和亚硝酸盐水平。
主要结果
LPS 刺激诱导大鼠腹膜巨噬细胞产生促炎表型。Flavocoxid（128 μg·mL-1）显著抑制 COX-2（LPS = 18 ± 2.1；flavocoxid = 3.8 ± 0.9 积分强度）、5-LOX（LPS = 20 ± 3.8；flavocoxid = 3.1 ± 0.8 积分强度）和 iNOS 表达（LPS = 15 ± 1.1；flavocoxid = 4.1 ± 0.4 积分强度），但不改变 COX-1 表达。因此，培养上清液中的 PGE2 和 LTB4 水平降低。Flavocoxid 还防止了 IkappaB-α 蛋白的丢失（LPS = 1.9 ± 0.2；flavocoxid = 7.2 ± 1.6 积分强度），减弱了 LPS 诱导的 NF-kappaB 结合活性的增加（LPS = 9.2 ± 2；flavocoxid = 2.4 ± 0.7 积分强度），并降低了 LPS 诱导的 TNF-α mRNA 水平（LPS = 8 ± 0.9；flavocoxid = 1.9 ± 0.8 倍/β-肌动蛋白）。最后，Flavocoxid 降低了 LPS 刺激的巨噬细胞中的 MDA、TNF 和亚硝酸盐水平。
结论和意义
Flavocoxid 可能作为一种潜在的抗炎剂，在基因和蛋白表达水平上发挥作用。
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