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摘要：目的：研究 Apigenin glucuronoside抑制巨噬细胞的分子机制。方法：通过检测LPS刺激RAW264.7细胞释放的TNF-、PGE2 、NO等炎症介质，评价药物对巨噬细胞释放炎症介质的抑制作用。以Griess法检测NO含量，以酶免疫测定试剂盒检测TNF-α、PGE2含量，同时以MTT法评价细胞毒性。半定量RT-PCR法检测炎症介质基因mRNA表达量；Western blot法检测IkBa、p-IkBa、p-ERK、ERK、p-JNK、JNK、p-p38、p38及内参蛋白β-actin水平；以乙醇诱导小鼠胃溃疡模型进行体内试验验证药物抗炎效果。结果：AGG可以抑制炎性介质在巨噬细胞中的表达，并抑制相关炎症基因mRNA的表达。AGG主要抑制MAPKs信号通路中ERK和p38的表达量。体内试验表明，AGG可改善乙醇/盐酸诱发的小鼠胃炎损伤情况。 结论：AGG可能通过抑制ERK和p38激酶的磷酸化，从而抑制LPS所诱导的炎症因子的转录，从而抑制炎症反应。54006
关键词：杜仲，Apigenin glucuronoside，巨噬细胞，脂多糖，信号通路
Abstract:【Objective】To study the molecular mechanism of Apigenin glucuronoside in LPS-stimulated macrophage model. the inhibition .【Method】RAW264.7 cells can be activated by LPS and release inflammatory mediators, such as tumor necrosis factor (TNF)-, nitric oxide (NO) and prostaglandin E2 (PGE2) . NO production was determined by Griess assay and the production of TNF-α、PGE2 was determined by an enzyme immunoassay kit. Moreover, cell viability was determined by MTT assay. Semi-quantitative RT-PCR was used to test the levels of mRNA expression of inflammatory mediators. The levels of IB、p-IB、p-ERK、ERK、p-JNK、JNK、p-p38、p38 and-actin were examined by western blot. In vivo experiment in accordance with the EtOH/HCl-induced gastritis mouse model was applied to test the anti-inflammatory effects of AGG. 【Result】AGG suppresses the production of inflammatory mediators and the mRNA expression of inflammatory genes in macrophages. AGG could suppress the activities of ERK and p38 in MAPK pathways. AGG ameliorates EtOH/HCl-Induced gastritis in animal disease models. 【Conclusion】AGG may inhibit the phosphorylation of ERK and p38, thereby inhibiting the transcription of inflammatory factors induced by LPS , resulting in inhibiting the inflammatory reaction.
Key word: Eucommia ulmoides Oliver, apigenin glucuronoside, macrophage cell, lipopolysaccharide (LPS), signal transduction
目录
1 文献综述 4
1.1杜仲 4
1.1.1 杜仲的药理作用与研究进展 4
1.1.2 杜仲中提取的药物单体Apigenin glucuronoside 4
1.2炎症 4
1.2.1 炎症反应 4
1.1.2 巨噬细胞 5
1.3 LPS诱导的炎症信号传导机制 5
1.3.1 细菌内毒素性致病 5
1.3.2 LPS及其诱导的信号通路 5
2 方法与材料 6
2.1 试剂与仪器 6
2.2 小鼠 6
2.3 细胞培养及传代 6
2.4 NO、PGE2和TNF-a表达量的测定 7
2.5 细胞体外生长活性检测 7
2.6半定量逆转录酶（RT）聚合酶链反应进行基因分析 7
2.7制备细胞裂解液及细胞核提取液并用Western印迹进行分析