【摘要】：目的：本試驗(yàn)采用傳統(tǒng)中藥黃芪的主要成分黃芪總黃酮為研究對(duì)象,探討了黃芪總黃酮的體外抗炎免疫作用及其相關(guān)作用機(jī)制。方法：以正常條件下和脂多糖(LPS)誘導(dǎo)下的小鼠腹腔巨噬細(xì)胞RAW264.7為模型,分別設(shè)立空白組和藥物低、中、高劑量組。通過細(xì)胞毒性測定,擬定黃芪總黃酮的試驗(yàn)劑量；通過Griess試劑盒測定NO表達(dá)變化,通過ELISA試劑盒檢測因子IL-6、IL-1β、IFN-γ、TNF-α和介質(zhì)PGE2等的變化規(guī)律；使用RT-PCR和凝膠電泳方法測定小鼠腹腔巨噬細(xì)胞RAW 264.7細(xì)胞COX-2 mRNA和iNOS mRNA的含量變化；采用Western blot法分析iNOS和COX-2蛋白含量變化,同時(shí)檢測MAPKs信號(hào)通路的關(guān)鍵蛋白p38、ERK1/2和JNK磷酸化水平變化。結(jié)果：1.黃芪總黃酮低、中劑量組在一定程度上上調(diào)了正常條件下RAW264.7小鼠腹腔巨噬細(xì)胞上清液中NO、PGE2和IL-6、 IL-1β、IFN-γ、rNF-α的表達(dá)水平；黃芪總黃酮?jiǎng)┝拷M具有下調(diào)LPS誘導(dǎo)后的RAW 264.7小鼠腹腔巨噬細(xì)胞上清液中NO、PGE2和IL-6、IL-1β、IFN-γ、TNF-α的過度表達(dá)作用。2.黃芪總黃酮各劑量組分別不同程度的上調(diào)了正常條件下細(xì)胞iNOS和COX-2 mRNA的轉(zhuǎn)錄水平,并抑制了LPS誘導(dǎo)后的iNOS和COX-2 mRNA轉(zhuǎn)錄水平,且滿足一定程度上的劑量依賴關(guān)系。3.低劑量組黃芪總黃酮在一定程度上上調(diào)了小鼠腹腔巨噬細(xì)胞RAW264.7iNOS和COX-2蛋白表達(dá)以及通路蛋白p38和JNK激酶的磷酸化水平,各劑量黃芪總黃酮能顯著的抑制LPS誘導(dǎo)后iNOS和COX-2的蛋白以及通路蛋白p38和.JNK激酶的磷酸化,并呈劑量依賴關(guān)系,對(duì)ERK的磷酸化水平無明顯作用。結(jié)論：研究表明,黃芪總黃酮通過調(diào)控正常條件下和LPS誘導(dǎo)的小鼠RAW 264.7細(xì)胞ERK 1/2和p38 MAPK信號(hào)傳導(dǎo)通路以及iNOS和COX-2mRNA和蛋白表達(dá),從而調(diào)控NO、PGE2和IL-6、IL-1β、IFN-γ、TNF-α的生成,發(fā)揮其體外抗炎免疫雙向調(diào)節(jié)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- 緯, TNF- 偽 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- 緯, rNF- 偽 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- 緯, TNF- 偽 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- 緯 and TNF- 偽 plays a bidirectional role in anti-inflammatory immunity in vitro.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S853.7

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本文編號(hào)：2335214