橙皮素对视网膜色素上皮细胞氧化应激和炎症的保护作用及机制

发布时间：2018-04-17 13:02

本文选题：橙皮素 + 氧化应激　；参考：《吉林大学》2015年博士论文

【摘要】：本研究首先利用t-BHP和LPS诱导RAW264.7细胞建立氧化应激和炎症反应的模型，初步探究橙皮素的抗氧化和抗炎作用。然后，采用H2O2诱导ARPE-19细胞产生氧化应激和炎症反应，观察橙皮素对其保护作用并分析其相关分子机制，以及橙皮素在小鼠原代RPE细胞中的保护作用，对预防和治疗年龄相关性黄斑变性（AMD）提供新的策略。本实验应用酶联免疫吸附实验（ELISA）、免疫印迹法（Western Blot）、逆转录聚合酶链反应（RT-PCR）、流式细胞术（FCM）和免疫共沉淀（Co-IP）等技术，从细胞、分子以及信号转导水平等方面对橙皮素的抗氧化和抗炎作用机制进行研究。结果如下：（1）实验（一）结果表明：在RAW264.7细胞中，橙皮素不仅能通过激活抗氧化Keap1/Nrf2/ARE信号通路，上调抗氧化酶HO-1、NQO1和GST表达，抑制t-BHP诱导的细胞活性下降和ROS的产生，而且能通过抑制MAPK和NF-κB信号通路激活，降低LPS诱导的TNF-α、IL-6、IL-1β、iNOS和COX-2等炎性介质的表达。（2）RPE细胞受到氧化应激和炎症损伤是AMD发病和进展的一个重要因素，因此在第一部分实验结果的基础上，我们进一步探究橙皮素对H2O2诱导的ARPE-19细胞氧化应激和炎症损伤的保护作用。实验（二）结果表明：橙皮素不仅能通过增加抗氧化因子HO-1、GCLC和GCLM的表达，抑制H2O2诱导的SOD和GSH的消耗以及MDA的产生而发挥抗氧化作用，并且能通过抑制MAPK和NF-κB信号通路的激活，降低TNF-α、IL-1β、IL-18和IL-6的产生而起到抗炎效果。因此，橙皮素具有抗氧化抗炎的双重活性，从而能抑制H2O2引起的细胞活性下降、细胞调亡以及ROS的产生，进而对ARPE-19细胞产生保护作用。但是，橙皮素抑制H2O2诱导的细胞活性下降、ROS产生以及炎性因子产生的作用可被HO-1抑制剂SnPP逆转。（3）实验（三）结果表明：橙皮素能诱导Keap1/Nrf2/ARE信号通路及其调控HO-1蛋白表达；在H2O2不激活Nrf2和HO-1的表达但对Txnip、NLRP3和Caspase-1有激活作用的条件下，，橙皮素能显著抑制H2O2诱导的Txnip、IL-1β和IL-18的表达，抑制NLRP3与Caspase1、ASC相互结合以及Txnip与NLRP3的相互结合，并增强了Trx表达及其与Txnip的相互结合，而对NLRP3与Caspase1、ASC的表达无直接影响作用，但橙皮素这些作用，可被SnPP逆转。最后，橙皮素在原代RPE细胞中也能够显著诱导Nrf2/HO-1蛋白的表达并抑制H2O2诱导的原代RPE细胞的死亡。综上所述，本研究首次阐明了橙皮素能够通过抑制H2O2诱导的细胞活性下降、ROS和炎性因子的产生而发挥对ARPE-19细胞损伤的保护作用。这主要是通过橙皮素激活Nrf2调控HO-1的表达，而抑制Txnip的活化、Txnip与NLRP3的相互结合以及NLRP3炎性小体的激活而发挥作用。因此，以激活Nrf2/HO-1为标靶，橙皮素发挥抗氧化和抗炎活性，有望成为防治年龄相关性黄斑变性（AMD）的一个新途径。
[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- 伪, 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).
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.5

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