安石榴苷通过诱导小鼠巨噬细胞M2型分化抑制急性炎症反应

发布时间：2018-04-16 15:14

本文选题：安石榴苷 + 小鼠　；参考：《中国农业大学》2015年博士论文

【摘要】：安石榴苷是石榴皮提取物中的有效成分,在石榴皮发挥抗炎和抗氧化效果中起到关键作用。本文通过研究安石榴苷抑制巨噬细胞炎症反应,并促进巨噬细胞向M2型转化的分子机制,为安石榴苷的临床应用提供理论依据和技术支持。试验一本试验首先采用小鼠单核-巨噬细胞系RAW264.7作为体外研究对象,探讨安石榴苷对脂多糖(lipopolysaccharide, LPS)诱导炎症反应的调节作用及分子机制。通过Griess反应、ELISA、 RT-PCR和Western Blot等方法检测发现,安石榴苷可以显著抑制LPS诱导的巨噬细胞一氧化氮(Nitric oxide, NO)、前列腺素E2(Prostin E2, PGE2)、白介素(interleukin,IL)-1β、IL-6和肿瘤坏死因子α(tumor necrosis factor α, TNF-α)的生成,缓解LPS诱导的过度炎症反应进程。机制研究发现,安石榴苷可以显著抑制MAPKs家族蛋白p38、JNK、ERK1/2的磷酸化水平,并通过降低IκBα的降解达到抑制NF-κB亚基p65磷酸化。试验二本试验研究安石榴苷在RAW264.7巨噬细胞中发挥的抗氧化作用及分子机制。结果显示,安石榴苷可以激活巨噬细胞内Nrf2和血红素加氧酶1(heme oxygenase, HO-1)蛋白水平的表达,同时上调Akt蛋白的磷酸化水平。但加入PI3K/Akt抑制剂LY294002后,安石榴苷激活HO-1蛋白的能力受到显著抑制,表明PI3K/Akt在安石榴苷激活HO-1过程中发挥重要的作用。此外,安石榴苷可以显著降低LPS诱导的巨噬细胞内氧自由基的生成,并对超氧化物歧化酶1(Superoxide dismutase1,SOD1)活性有明显调节作用,提示安石榴苷可以通过激抑制巨噬细胞内氧化应激降低LPS诱导的巨噬细胞炎症反应。试验三本试验利用LPS诱导M1型巨噬细胞分化,进而通过流式细胞术和Western Blot等方法测定安石榴苷对巨噬细胞分型特异指标的调节作用。结果显示,安石榴苷可以显著降低LPS诱导的M1型巨噬细胞表面CD11c+的生成,增加CD206的数量。同时,安石榴苷可以显著降低iNOS蛋白的生成,提高Arg-1蛋白表达,促进抗炎通路STAT-3/IL-10的激活。进一步考察发现,加入HO-1蛋白抑制剂ZnPP后,安石榴苷诱导HO-1表达上调的作用被显著抑制,其促进巨噬细胞向M2型转化的作用也被抑制,表明HO-1在安石榴苷诱导巨噬细胞由M1型向M2型转化的进程中发挥主要调节作用。试验四本试验为了验证安石榴苷在小鼠原代细胞中的抗炎作用及调控机制,我们采用小鼠腹腔巨噬细胞进行相关试验。结果显示,安石榴苷可以显著降低小鼠腹腔巨噬细胞M1巨噬细胞表面标记CD11c+含量,提高M2型巨噬细胞表面标记CD206的数量,抑制iNOS蛋白的表达,上调Arg-1蛋白,且通过STAT-3/IL-10/HO-1信号通路调节M2型巨噬细胞分化,与体外结果基本致。
[Abstract]:Pomegranate glucoside is an effective component in the extract of pomegranate peel, which plays a key role in the anti-inflammatory and anti-oxidation effects of pomegranate peel.In this paper, we studied the molecular mechanism of the inhibition of macrophage inflammation and the transformation of macrophages to M2 type, which provided the theoretical basis and technical support for the clinical application of amgranate.Test oneIn this experiment, mouse monocyte-macrophage cell line RAW264.7 was used as an in vitro study to investigate the modulatory effect and molecular mechanism of ampoulidine on lipopolysaccharide (LPS) -induced inflammation.By Griess reaction, RT-PCR and Western Blot, it was found that amomegranate could significantly inhibit the production of nitric oxide nitric oxide, NOX, prostaglandin E2(Prostin E2, PGE2, interleukin-interleukin-1 尾 -guan6, and tumor necrosis factor 伪 -tumor necrosis factor 伪 (TNF- 伪) induced by LPS.Alleviate the process of excessive inflammation induced by LPS.It was found that amomegranate could significantly inhibit the phosphorylation of MAPKs family protein p38, and inhibit the phosphorylation of NF- 魏 B subunit p65 by decreasing the degradation of I 魏 B 伪.Test IIThe antioxidation and molecular mechanism of amgranate in RAW264.7 macrophages were studied.The results showed that amomegranate could activate the expression of Nrf2 and heme oxygenase 1(heme oxygenase (HO-1) in macrophages and up-regulate the phosphorylation of Akt protein.However, the ability of engranate to activate HO-1 protein was significantly inhibited by the addition of PI3K/Akt inhibitor LY294002, indicating that PI3K/Akt plays an important role in the activation of HO-1 by amomegranate.In addition, garnet could significantly reduce the production of oxygen free radicals in macrophages induced by LPS and regulate the activity of superoxide dismutase 1(Superoxide dismutase 1 (SOD1).These results suggest that amgranate can reduce the inflammatory response of macrophages induced by LPS by inhibiting oxidative stress in macrophages.Test IIIIn this experiment, LPS was used to induce the differentiation of M1 type macrophages, and then the regulation of garnet glucoside on specific indexes of macrophage typing was determined by flow cytometry and Western Blot.The results showed that pomegranate could significantly reduce the formation of CD11c on the surface of M1 macrophages induced by LPS and increase the number of CD206.At the same time, pomegranate can significantly reduce the production of iNOS protein, increase the expression of Arg-1 protein, and promote the activation of STAT-3/IL-10 in anti-inflammatory pathway.It was found that the upregulation of HO-1 expression induced by amgranate was significantly inhibited after the addition of HO-1 protein inhibitor ZnPP, and the effect on macrophage transformation to M2 type was also inhibited.These results suggest that HO-1 plays a major regulatory role in the process of macrophage transformation from M 1 to M 2 induced by garnet glucoside.Test IVIn order to verify the anti-inflammatory effect and regulation mechanism of garnet glucoside in primary mouse cells, we used mouse peritoneal macrophages to carry out the related tests.The results showed that garnet could significantly decrease the CD11c content on macrophages of peritoneal macrophages M1, increase the number of labeled CD206 on M 2 macrophages, inhibit the expression of iNOS protein, and up-regulate Arg-1 protein.The differentiation of M 2 macrophages was regulated by STAT-3/IL-10/HO-1 signaling pathway.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S853.74

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