黄芪总黄酮体外抗炎免疫双向调节作用及相关机制研究

发布时间：2018-11-16 10:04

【摘要】：目的：本试验采用传统中药黄芪的主要成分黄芪总黄酮为研究对象,探讨了黄芪总黄酮的体外抗炎免疫作用及其相关作用机制。方法：以正常条件下和脂多糖(LPS)诱导下的小鼠腹腔巨噬细胞RAW264.7为模型,分别设立空白组和药物低、中、高剂量组。通过细胞毒性测定,拟定黄芪总黄酮的试验剂量；通过Griess试剂盒测定NO表达变化,通过ELISA试剂盒检测因子IL-6、IL-1β、IFN-γ、TNF-α和介质PGE2等的变化规律；使用RT-PCR和凝胶电泳方法测定小鼠腹腔巨噬细胞RAW 264.7细胞COX-2 mRNA和iNOS mRNA的含量变化；采用Western blot法分析iNOS和COX-2蛋白含量变化,同时检测MAPKs信号通路的关键蛋白p38、ERK1/2和JNK磷酸化水平变化。结果：1.黄芪总黄酮低、中剂量组在一定程度上上调了正常条件下RAW264.7小鼠腹腔巨噬细胞上清液中NO、PGE2和IL-6、 IL-1β、IFN-γ、rNF-α的表达水平；黄芪总黄酮剂量组具有下调LPS诱导后的RAW 264.7小鼠腹腔巨噬细胞上清液中NO、PGE2和IL-6、IL-1β、IFN-γ、TNF-α的过度表达作用。2.黄芪总黄酮各剂量组分别不同程度的上调了正常条件下细胞iNOS和COX-2 mRNA的转录水平,并抑制了LPS诱导后的iNOS和COX-2 mRNA转录水平,且满足一定程度上的剂量依赖关系。3.低剂量组黄芪总黄酮在一定程度上上调了小鼠腹腔巨噬细胞RAW264.7iNOS和COX-2蛋白表达以及通路蛋白p38和JNK激酶的磷酸化水平,各剂量黄芪总黄酮能显著的抑制LPS诱导后iNOS和COX-2的蛋白以及通路蛋白p38和.JNK激酶的磷酸化,并呈剂量依赖关系,对ERK的磷酸化水平无明显作用。结论：研究表明,黄芪总黄酮通过调控正常条件下和LPS诱导的小鼠RAW 264.7细胞ERK 1/2和p38 MAPK信号传导通路以及iNOS和COX-2mRNA和蛋白表达,从而调控NO、PGE2和IL-6、IL-1β、IFN-γ、TNF-α的生成,发挥其体外抗炎免疫双向调节作用。
[Abstract]:Aim: to investigate the anti-inflammatory and immunological effects of total flavonoids of Astragalus membranaceus in vitro. Methods: normal and lipopolysaccharide (LPS) induced mouse peritoneal macrophages (RAW264.7) were used as models, and blank group and low, middle and high dose groups were set up respectively. The experimental dose of total flavonoids of Astragalus membranaceus was determined by cytotoxicity test, the expression of NO was detected by Griess kit, and the changes of IL-6,IL-1 尾, IFN- 纬, TNF- 伪 and medium PGE2 were detected by ELISA kit. The contents of COX-2 mRNA and iNOS mRNA in RAW 264.7 cells of mouse peritoneal macrophages were measured by RT-PCR and gel electrophoresis. The changes of iNOS and COX-2 protein contents were analyzed by Western blot, and the phosphorylation levels of p38 ERK1 / 2 and JNK in MAPKs signaling pathway were detected simultaneously. Results: 1. In the middle dose group, the expression of NO,PGE2, IL-6, IL-1 尾, IFN- 纬, rNF- 伪 in the peritoneal macrophage supernatant of RAW264.7 mice was up-regulated to some extent. Total flavonoids of Astragalus membranaceus could down-regulate the overexpression of NO,PGE2 and IL-6,IL-1 尾, IFN- 纬, TNF- 伪 in the supernatant of peritoneal macrophages induced by LPS in RAW 264.7 mice. 2. The total flavonoids of Astragalus membranaceus upregulated the transcription levels of iNOS and COX-2 mRNA in normal condition, and inhibited the transcription of iNOS and COX-2 mRNA induced by LPS, and satisfied the dose-dependent relationship to a certain extent. In low dose group, the total flavonoids of Astragalus membranaceus upregulated the expression of RAW264.7iNOS and COX-2 protein and the phosphorylation of p38 and JNK kinase in mouse peritoneal macrophages. The total flavonoids of Astragalus membranaceus could significantly inhibit the phosphorylation of iNOS and COX-2 proteins and pathway proteins p38 and. JNK kinases induced by LPS in a dose-dependent manner, but had no effect on the phosphorylation level of ERK. Conclusion: total flavonoids of Astragalus membranaceus can regulate the expression of NO,PGE2 and IL-6,IL-1 尾 by regulating the signal transduction pathways of ERK 1 / 2 and p38 MAPK, iNOS, COX-2mRNA and protein in RAW 264.7 cells induced by normal condition and LPS. The production of IFN- 纬 and TNF- 伪 plays a bidirectional role in anti-inflammatory immunity in vitro.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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