本文選題：安石榴苷 + 小鼠��； 參考：《中國農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：安石榴苷是石榴皮提取物中的有效成分,在石榴皮發(fā)揮抗炎和抗氧化效果中起到關(guān)鍵作用。本文通過研究安石榴苷抑制巨噬細(xì)胞炎癥反應(yīng),并促進(jìn)巨噬細(xì)胞向M2型轉(zhuǎn)化的分子機(jī)制,為安石榴苷的臨床應(yīng)用提供理論依據(jù)和技術(shù)支持。 試驗(yàn)一 本試驗(yàn)首先采用小鼠單核-巨噬細(xì)胞系RAW264.7作為體外研究對象,探討安石榴苷對脂多糖(lipopolysaccharide, LPS)誘導(dǎo)炎癥反應(yīng)的調(diào)節(jié)作用及分子機(jī)制。通過Griess反應(yīng)、ELISA、 RT-PCR和Western Blot等方法檢測發(fā)現(xiàn),安石榴苷可以顯著抑制LPS誘導(dǎo)的巨噬細(xì)胞一氧化氮(Nitric oxide, NO)、前列腺素E2(Prostin E2, PGE2)、白介素(interleukin,IL)-1β、IL-6和腫瘤壞死因子α(tumor necrosis factor α, TNF-α)的生成,緩解LPS誘導(dǎo)的過度炎癥反應(yīng)進(jìn)程。機(jī)制研究發(fā)現(xiàn),安石榴苷可以顯著抑制MAPKs家族蛋白p38、JNK、ERK1/2的磷酸化水平,并通過降低IκBα的降解達(dá)到抑制NF-κB亞基p65磷酸化。 試驗(yàn)二 本試驗(yàn)研究安石榴苷在RAW264.7巨噬細(xì)胞中發(fā)揮的抗氧化作用及分子機(jī)制。結(jié)果顯示,安石榴苷可以激活巨噬細(xì)胞內(nèi)Nrf2和血紅素加氧酶1(heme oxygenase, HO-1)蛋白水平的表達(dá),同時(shí)上調(diào)Akt蛋白的磷酸化水平。但加入PI3K/Akt抑制劑LY294002后,安石榴苷激活HO-1蛋白的能力受到顯著抑制,表明PI3K/Akt在安石榴苷激活HO-1過程中發(fā)揮重要的作用。此外,安石榴苷可以顯著降低LPS誘導(dǎo)的巨噬細(xì)胞內(nèi)氧自由基的生成,并對超氧化物歧化酶1(Superoxide dismutase1,SOD1)活性有明顯調(diào)節(jié)作用,提示安石榴苷可以通過激抑制巨噬細(xì)胞內(nèi)氧化應(yīng)激降低LPS誘導(dǎo)的巨噬細(xì)胞炎癥反應(yīng)。 試驗(yàn)三 本試驗(yàn)利用LPS誘導(dǎo)M1型巨噬細(xì)胞分化,進(jìn)而通過流式細(xì)胞術(shù)和Western Blot等方法測定安石榴苷對巨噬細(xì)胞分型特異指標(biāo)的調(diào)節(jié)作用。結(jié)果顯示,安石榴苷可以顯著降低LPS誘導(dǎo)的M1型巨噬細(xì)胞表面CD11c+的生成,增加CD206的數(shù)量。同時(shí),安石榴苷可以顯著降低iNOS蛋白的生成,提高Arg-1蛋白表達(dá),促進(jìn)抗炎通路STAT-3/IL-10的激活。進(jìn)一步考察發(fā)現(xiàn),加入HO-1蛋白抑制劑ZnPP后,安石榴苷誘導(dǎo)HO-1表達(dá)上調(diào)的作用被顯著抑制,其促進(jìn)巨噬細(xì)胞向M2型轉(zhuǎn)化的作用也被抑制,表明HO-1在安石榴苷誘導(dǎo)巨噬細(xì)胞由M1型向M2型轉(zhuǎn)化的進(jìn)程中發(fā)揮主要調(diào)節(jié)作用。 試驗(yàn)四 本試驗(yàn)為了驗(yàn)證安石榴苷在小鼠原代細(xì)胞中的抗炎作用及調(diào)控機(jī)制,我們采用小鼠腹腔巨噬細(xì)胞進(jìn)行相關(guān)試驗(yàn)。結(jié)果顯示,安石榴苷可以顯著降低小鼠腹腔巨噬細(xì)胞M1巨噬細(xì)胞表面標(biāo)記CD11c+含量,提高M(jìn)2型巨噬細(xì)胞表面標(biāo)記CD206的數(shù)量,抑制iNOS蛋白的表達(dá),上調(diào)Arg-1蛋白,且通過STAT-3/IL-10/HO-1信號通路調(diào)節(jié)M2型巨噬細(xì)胞分化,與體外結(jié)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 偽 -tumor necrosis factor 偽 (TNF- 偽) 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 偽.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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S853.74

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