β-胡萝卜素对脂多糖诱导巨噬细胞的炎症因子及NF-κB信号通路的影响

发布时间：2018-04-22 04:05

本文选题：β-胡萝卜素 + RAW264.7细胞　；参考：《吉林农业大学》2017年硕士论文

【摘要】：炎症反应的发生发展与断奶仔猪的肠道炎症以及多种疾病的产生密切相关,而甾体和非甾体这两种传统的抗炎物质在缓解炎症的同时存在极大的副作用,所以,寻求一种新型、高效、副作用小的缓解炎症的物质很有意义。作为一种天然类胡萝卜素,β-胡萝卜素是重要的维生素A源,具有抗氧化、促进细胞间隙连接通讯、提高免疫力等多种生物学功能。而NF-κB是炎症中一个重要的核转录因子,在受到脂多糖的刺激时,会诱导相关炎症因子的释放。所以,本研究以脂多糖诱导的巨噬细胞为炎症模型,探讨β-胡萝卜素对其炎症因子的作用以及与NF-κB通路的关系。本研究首先用MTT法检测细胞活性,确定β-胡萝卜素、LPS的最佳添加浓度和时间;荧光定量PCR、ELISA检测主要炎症因子IL-1β、IL-6、TNF-α的mRNA相对表达量和在细胞中的含量,建立细胞炎症模型,分析β-胡萝卜素对炎症因子的影响。然后,荧光定量PCR、Western Blot检测NF-κB p65 mRNA、蛋白的相对表达量,分析β-胡萝卜素对NF-κB通路的影响。最后,综合运用以上方法,分析LPS+PDTC+β-胡萝卜素和LPS+PDTC组对炎症因子、NF-κB p65的影响。结果如下:在β-胡萝卜素对炎症因子的研究中,发现β-胡萝卜素最佳添加浓度梯度和时间分别为20、40、80、160μmol/L和3 h,LPS最佳添加浓度和时间分别为5μg/mL和24 h。5μg/mL的LPS诱导巨噬细胞24 h后,用不同浓度的β-胡萝卜素处理细胞3 h可极显著降低炎症因子IL-1β、IL-6、TNF-α的mRNA相对表达量(P0.01);对炎症因子在细胞中的含量有显著降低的趋势,提示β-胡萝卜素对LPS诱导的巨噬细胞的炎症因子有抑制作用。在β-胡萝卜素对NF-κB通路的研究中,发现PDTC与LPS共同处理巨噬细胞24 h后,与LPS组相比,可显著降低NF-κB p65基因、蛋白的相对表达量(P0.05),极显著降低炎症因子IL-1β、IL-6、TNF-α的mRNA相对表达量(P0.01),减少炎症因子在细胞中的含量,提示在LPS诱导的巨噬细胞中,抑制NF-κB p65的表达可以抑制炎症因子。在NF-κB通路的特异性研究中,发现不同浓度的β-胡萝卜素可显著降低LPS诱导的NF-κB p65基因、蛋白的相对表达量(P0.05),说明在LPS诱导的巨噬细胞中,β-胡萝卜素通过抑制NF-κB p65的表达可以抑制炎症因子;LPS+PDTC+β-胡萝卜素组对炎症因子的抑制作用比LPS+PDTC组明显,但两组对NF-κB p65的表达差异不显著(P0.05),提示NF-κB通路不是β-胡萝卜素抑制炎症因子的特异性的通路。依据结果分析归纳总结,本研究发现β-胡萝卜素对LPS诱导的巨噬细胞的炎症因子有抑制作用,此抑制作用与NF-κB通路密切相关。
[Abstract]:The occurrence and development of inflammatory reaction is closely related to the intestinal inflammation of weaning piglets and the production of many diseases, while the two traditional anti-inflammatory substances, steroidal and non-steroidal, have great side effects while alleviating inflammation, so we seek a new type of inflammation. Highly effective, with little side effects, anti-inflammatory substances are significant. As a kind of natural carotenoid, 尾 -carotene is an important vitamin A source, which has many biological functions, such as anti-oxidation, promoting intercellular junctional communication, enhancing immunity and so on. NF- 魏 B is an important nuclear transcription factor in inflammation. When stimulated by lipopolysaccharide, NF- 魏 B induces the release of related inflammatory factors. In this study, macrophages induced by lipopolysaccharide were used as inflammatory model to investigate the effect of 尾 -carotene on inflammatory cytokines and the relationship between 尾 -carotene and NF- 魏 B pathway. In this study, MTT assay was used to determine the optimal concentration and time of 尾 -carotene lipopolysaccharide (尾 -carotene), and the relative expression of mRNA and the content of TNF- 伪 in cells were detected by fluorescence quantitative PCR Elisa to establish a cellular inflammatory model. To analyze the effect of 尾-carotene on inflammatory factors. Then, the relative expression of NF- 魏 B p65 mRNAs and the effect of 尾 -carotene on NF- 魏 B pathway were analyzed by fluorescence quantitative Blot. Finally, the effects of LPS PDTC 尾 -carotene and LPS PDTC on the inflammatory factor NF- 魏 B p65 were analyzed. The results were as follows: in the study of inflammatory factors induced by 尾 -carotene, it was found that the optimal concentration gradient and time of 尾 -carotene were 2040 mol/L and 80160 渭 mol/L, respectively, and the optimal concentration and time of LPS were 5 渭 g/mL and 24h.5 渭 g/mL, respectively. Treated with different concentrations of 尾 -carotene for 3 h, the relative expression of IL-1 尾 -IL-6TNF- 伪 mRNA was significantly decreased, and the content of inflammatory factors in the cells was significantly decreased. These results suggest that 尾-carotene inhibits the inflammatory cytokines of macrophages induced by LPS. In the study of 尾 -carotene on NF- 魏 B pathway, it was found that after co-treatment of PDTC and LPS for 24 h, NF- 魏 B p65 gene was significantly decreased compared with LPS group. The relative expression of P0.05 protein significantly decreased the relative expression of IL-1 尾 -IL-6TNF- 伪 mRNA and decreased the content of inflammatory factors in the cells. It suggested that the inhibition of NF- 魏 B p65 expression in macrophages induced by LPS could inhibit the expression of inflammatory factors. In the specific study of NF- 魏 B pathway, it was found that 尾 -carotene at different concentrations could significantly reduce the NF- 魏 B p65 gene induced by LPS. The relative expression of P0.05 protein indicated that 尾 -carotene could inhibit the expression of NF- 魏 B p65 by inhibiting the expression of NF- 魏 B p65 in macrophages induced by LPS. The inhibitory effect of 尾 -carotene on inflammatory cytokines in LPs PDTC 尾 -carotene group was more obvious than that in LPS PDTC group. However, there was no significant difference in the expression of NF- 魏 B p65 between the two groups, suggesting that the NF- 魏 B pathway was not a specific pathway for 尾 -carotene to inhibit inflammatory factors. According to the results, it was found that 尾 -carotene inhibited the inflammatory cytokines of macrophages induced by LPS, which was closely related to the NF- 魏 B pathway.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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