荔枝落果中A型低聚原花青素抑制类甜蛋白促炎机制

Inhibitory Mechanism of A-type Oligomeric Proanthocyanidins from Abscising Litchi Fruits on Proinflammation Induced by Litchi Thaumatin-Like Protein

以荔枝落果中的A型低聚原花青素为研究对象,探讨其对荔枝类甜蛋白(litchi thaumatin-like protein,LcTLP)促炎的抑制机理。采用乙醇提取、大孔吸附树脂纯化以及制备液相色谱仪等从荔枝落果中分离得到A型低聚原花青素(A-type oligomeric proanthocyanidins,A-OPC)纯化物,并对A-OPC结构鉴定,主要成分为原花青素A2、2个A型原花青素三聚体和1个A型四聚体,纯度达88.69%。利用LcTLP诱导构建RAW264.7细胞炎症模型,A-OPC的添加可抑制LcTLP诱导RAW264.7细胞的一氧化氮、细胞炎症因子白细胞介素-6和肿瘤坏死因子-α的分泌,在120μg/mL质量浓度下,抑制率分别可达58.38%、39.80%和86.31%(P<0.01)。蛋白免疫印迹分析发现,在120μg/mL质量浓度下A-OPC可降低诱导型一氧化氮合酶和环氧合酶2的表达量,并显著降低p65、核因子κB抑制蛋白、c-Jun氨基末端激酶、细胞外信号调节蛋白激酶和p38的磷酸化水平,说明A-OPC通过下调核转录因子-κB和丝裂原活化蛋白激酶通路中下游蛋白的磷酸化水平抑制LcTLP诱导的炎症反应。

This study investigated the mechanism by which A-type oligomeric proanthocyanidins(A-OPC)from abscising litchi fruits inhibit proinflammation induced by litchi thaumatin-like protein(LcTLP).A-OPC were prepared by sequential ethanol extraction and purification by macroporous adsorption resin and preparative liquid chromatography.Structural analysis showed that A-OPC were mainly composed of proanthocyanidin A2,two A-type proanthocyanidin trimers and one A-type tetramer,with a purity of 88.69%.At 120μg/mL concentration,A-OPC inhibited the secretion of nitric oxide(NO),interleukin(IL)-6 and tumor necrosis factor-α(TNF-α)by 58.38%,39.80%and 86.31%in RAW264.7 cells treated with LcTLP,respectively(P<0.01).Western blot showed that 120μg/mL A-OPC reduced the expression of inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2),and significantly decreased the phosphorylation levels of p65,inhibitor of NF-κB(IκBα),c-Jun N-terminal kinase(JNK),extracellular signal-regulated kinase(ERK)and p38.Therefore,A-OPC down-regulated the phosphorylation levels of downstream proteins in the nuclear transcription factor-κB(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways,thereby suppressing LcTLP-induced inflammatory response.