本文選題：橙皮素 + 氧化應(yīng)激��； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：本研究首先利用t-BHP和LPS誘導(dǎo)RAW264.7細(xì)胞建立氧化應(yīng)激和炎癥反應(yīng)的模型，初步探究橙皮素的抗氧化和抗炎作用。然后，采用H2O2誘導(dǎo)ARPE-19細(xì)胞產(chǎn)生氧化應(yīng)激和炎癥反應(yīng)，觀察橙皮素對(duì)其保護(hù)作用并分析其相關(guān)分子機(jī)制，以及橙皮素在小鼠原代RPE細(xì)胞中的保護(hù)作用，對(duì)預(yù)防和治療年齡相關(guān)性黃斑變性（AMD）提供新的策略。本實(shí)驗(yàn)應(yīng)用酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA）、免疫印跡法（Western Blot）、逆轉(zhuǎn)錄聚合酶鏈反應(yīng)（RT-PCR）、流式細(xì)胞術(shù)（FCM）和免疫共沉淀（Co-IP）等技術(shù)，從細(xì)胞、分子以及信號(hào)轉(zhuǎn)導(dǎo)水平等方面對(duì)橙皮素的抗氧化和抗炎作用機(jī)制進(jìn)行研究。結(jié)果如下： （1）實(shí)驗(yàn)（一）結(jié)果表明：在RAW264.7細(xì)胞中，橙皮素不僅能通過(guò)激活抗氧化Keap1/Nrf2/ARE信號(hào)通路，上調(diào)抗氧化酶HO-1、NQO1和GST表達(dá)，抑制t-BHP誘導(dǎo)的細(xì)胞活性下降和ROS的產(chǎn)生，而且能通過(guò)抑制MAPK和NF-κB信號(hào)通路激活，降低LPS誘導(dǎo)的TNF-α、IL-6、IL-1β、iNOS和COX-2等炎性介質(zhì)的表達(dá)。 （2）RPE細(xì)胞受到氧化應(yīng)激和炎癥損傷是AMD發(fā)病和進(jìn)展的一個(gè)重要因素，因此在第一部分實(shí)驗(yàn)結(jié)果的基礎(chǔ)上，我們進(jìn)一步探究橙皮素對(duì)H2O2誘導(dǎo)的ARPE-19細(xì)胞氧化應(yīng)激和炎癥損傷的保護(hù)作用。實(shí)驗(yàn)（二）結(jié)果表明：橙皮素不僅能通過(guò)增加抗氧化因子HO-1、GCLC和GCLM的表達(dá)，抑制H2O2誘導(dǎo)的SOD和GSH的消耗以及MDA的產(chǎn)生而發(fā)揮抗氧化作用，并且能通過(guò)抑制MAPK和NF-κB信號(hào)通路的激活，降低TNF-α、IL-1β、IL-18和IL-6的產(chǎn)生而起到抗炎效果。因此，橙皮素具有抗氧化抗炎的雙重活性，從而能抑制H2O2引起的細(xì)胞活性下降、細(xì)胞調(diào)亡以及ROS的產(chǎn)生，進(jìn)而對(duì)ARPE-19細(xì)胞產(chǎn)生保護(hù)作用。但是，橙皮素抑制H2O2誘導(dǎo)的細(xì)胞活性下降、ROS產(chǎn)生以及炎性因子產(chǎn)生的作用可被HO-1抑制劑SnPP逆轉(zhuǎn)。 （3）實(shí)驗(yàn)（三）結(jié)果表明：橙皮素能誘導(dǎo)Keap1/Nrf2/ARE信號(hào)通路及其調(diào)控HO-1蛋白表達(dá)；在H2O2不激活Nrf2和HO-1的表達(dá)但對(duì)Txnip、NLRP3和Caspase-1有激活作用的條件下，，橙皮素能顯著抑制H2O2誘導(dǎo)的Txnip、IL-1β和IL-18的表達(dá)，抑制NLRP3與Caspase1、ASC相互結(jié)合以及Txnip與NLRP3的相互結(jié)合，并增強(qiáng)了Trx表達(dá)及其與Txnip的相互結(jié)合，而對(duì)NLRP3與Caspase1、ASC的表達(dá)無(wú)直接影響作用，但橙皮素這些作用，可被SnPP逆轉(zhuǎn)。最后，橙皮素在原代RPE細(xì)胞中也能夠顯著誘導(dǎo)Nrf2/HO-1蛋白的表達(dá)并抑制H2O2誘導(dǎo)的原代RPE細(xì)胞的死亡。 綜上所述，本研究首次闡明了橙皮素能夠通過(guò)抑制H2O2誘導(dǎo)的細(xì)胞活性下降、ROS和炎性因子的產(chǎn)生而發(fā)揮對(duì)ARPE-19細(xì)胞損傷的保護(hù)作用。這主要是通過(guò)橙皮素激活Nrf2調(diào)控HO-1的表達(dá)，而抑制Txnip的活化、Txnip與NLRP3的相互結(jié)合以及NLRP3炎性小體的激活而發(fā)揮作用。因此，以激活Nrf2/HO-1為標(biāo)靶，橙皮素發(fā)揮抗氧化和抗炎活性，有望成為防治年齡相關(guān)性黃斑變性（AMD）的一個(gè)新途徑。
[Abstract]:In this study, the oxidative stress and inflammatory response of RAW264.7 cells induced by t-BHP and LPS were established to explore the antioxidant and anti-inflammatory effects of hesperidin.Then, ARPE-19 cells were induced by H2O2 to produce oxidative stress and inflammatory response. The protective effect of hesperidin on ARPE-19 cells was observed and its molecular mechanism was analyzed. The protective effect of hesperidin on primary RPE cells was also analyzed.To provide a new strategy for the prevention and treatment of age-related macular degeneration (AMDD).In this study, Elisa, Western blotl, reverse transcriptase polymerase chain reaction (RT-PCR), flow cytometry (FCM) and co-immunoprecipitation (Co-IPP) techniques were used to detect the expression of EISAA, Western blotl, reverse transcriptase polymerase chain reaction (RT-PCR), flow cytometry (FCM) and co-immunoprecipitation (Co-IP).The mechanism of anti-oxidation and anti-inflammatory effect of hesperidin was studied in molecular and signal transduction level.The results are as follows:The results showed that in RAW264.7 cells, hesperidin not only upregulated the expression of antioxidant enzyme HO-1NQO1 and GST, but also inhibited the decrease of cell activity and the production of ROS induced by t-BHP by activating the antioxidant Keap1/Nrf2/ARE signaling pathway.Moreover, it can inhibit the activation of MAPK and NF- 魏 B signaling pathway, and decrease the expression of inflammatory mediators such as TNF- 偽, IL-6, IL-1 尾, iNOS and COX-2 induced by LPS.Oxidative stress and inflammatory injury are an important factor in the pathogenesis and progression of AMD. Therefore, based on the results of the first part of the experiment,We further investigated the protective effects of hesperidin on oxidative stress and inflammatory injury induced by H2O2 in ARPE-19 cells.The results of experiment (2) showed that hesperidin not only inhibited the consumption of SOD and GSH induced by H2O2 and the production of MDA, but also inhibited the activation of MAPK and NF- 魏 B signaling pathway by increasing the expression of HO-1GCLC and GCLM.The anti-inflammatory effect was achieved by decreasing the production of IL-18 and IL-6.Therefore, hesperidin has the dual activity of anti-oxidation and anti-inflammation, which can inhibit the decrease of cell activity induced by H2O2, cell apoptosis and the production of ROS, and thus protect ARPE-19 cells.However, the inhibitory effect of hesperidin on the production of Ros and inflammatory factors induced by H2O2 could be reversed by HO-1 inhibitor SnPP.(3) the results of experiment (3) showed that hesperidin could induce Keap1/Nrf2/ARE signaling pathway and regulate the expression of HO-1 protein, and under the condition that H2O2 did not activate the expression of Nrf2 and HO-1, but could activate the expression of Nrf2 and Caspase-1, hesperidin could significantly inhibit the expression of IL-1 尾 and IL-18 induced by H2O2.Inhibition of the binding of NLRP3 and Caspase1A ASC and the combination of Txnip and NLRP3 enhanced the expression of Trx and its interaction with Txnip, but had no direct effect on the expression of NLRP3 and Caspase1 + ASC, but these effects of hesperidin could be reversed by SnPP.Finally, hesperidin could significantly induce the expression of Nrf2/HO-1 protein and inhibit the death of primary RPE cells induced by H2O2 in primary RPE cells.In conclusion, this study for the first time demonstrated that hesperidin can protect ARPE-19 cells by inhibiting the production of Ros and inflammatory factors induced by H2O2.It is mainly through the activation of Nrf2 by hesperidin to regulate the expression of HO-1, and to inhibit the binding of Txnip to NLRP3 and the activation of NLRP3 inflammatory corpuscles.Therefore, with activation of Nrf2/HO-1 as a target, hesperidin plays an important role in anti-oxidative and anti-inflammatory activities, which may be a new way to prevent and treat age-related macular degeneration (AMDD).
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R774.5

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