喹烯酮引起HepG2细胞程序性死亡的分子机制研究

发布时间：2018-06-18 21:50

本文选题：喹烯酮 + 凋亡　；参考：《中国农业大学》2016年博士论文

【摘要】：喹烯酮是我国自主研发的一类新兽药,其作为饲料添加剂广泛应用于畜牧水产养殖的生产实践中,具有良好的抗菌促生长作用。上世纪九十年代一系列的常规毒理试验证明了喹烯酮作为饲料添加剂的安全性。但是进入新世纪后,食品安全的重要性越来越受到关注。喹烯酮属于喹VA啉类化合物,同一类中还包括喹乙醇、卡巴氧等。喹乙醇和卡巴氧都被证实具有显著的毒性,与其拥有相同核心结构的喹烯酮的安全性也受到了重新审视。最近本实验室对喹烯酮引起的细胞凋亡及其通路进行了研究,对喹烯酮引起细胞的死亡机制有了初步了解。本研究拟从细胞程序性死亡的角度对喹烯酮作用下细胞死亡的分子机制做进一步的探索。试验结果表明喹烯酮与ML-7联用时,对HepG2细胞毒性增加,细胞凋亡率进一步升高,线粒体膜电位消失的效应增强,外源性和内源性凋亡通路都更加被激活。以上结果表明,喹烯酮通过凋亡引起细胞死亡的过程可以被ML-7加强。进一步研究表明,ML-7刺激了喹烯酮引起的MAPKs信号通路,同时抑制了喹烯酮引起的Akt信号通路。并且,运用MAPKs抑制剂和Akt激动剂之后的试验结果表明,Akt通络和MAPKs通路都参与了喹烯酮引起的细胞凋亡。综上所述,喹烯酮可以通过Akt通路和MAPKs通路调控凋亡的发生,具体机制为喹烯酮通过p38来诱发凋亡,而ERK, JNK和Akt通路则在此过程中抑制凋亡的发生。自噬又被称为细胞Ⅱ型程序性死亡,GFP-LC3质粒转染喹烯酮处理的HepG2细胞发现,喹烯酮使细胞中LC3聚集点增多,LC3的转化率呈喹烯酮浓度依赖性和时间依赖性升高。结果表明喹烯酮引起了HepG2细胞发生自噬。接着,研究发现内质网应激蛋白BiP和CHOP的表达量呈喹烯酮时间依赖性和浓度依赖性升高,并且sec24D、HerpUD、BiP的转录活性呈喹烯酮浓度依赖性升高。结果表明喹烯酮引起了HepG2细胞内的内质网应激。进一步研究发现,喹烯酮处理后,通过ATF6/DAPK1/MRLC的级联反应使mAtg9a的转移加强,从而促进了自噬小体的生成。在干扰掉ATF6和DAPK1后,自噬标志蛋白LC3的转化率随之降低。综上所述,喹烯酮通过引起内质网应激诱导了自噬通量的上升。线粒体自噬是一种具有底物特异性的选择性自噬。本研究结果表明,线粒体自噬的核心调控蛋白parkin和PINK1在喹烯酮作用的HepG2细胞中呈浓度依赖性和时间依赖性降低；。线粒体膜蛋白COX IV蛋白表达降低、Tom20和Tim23的蛋白表达升高,与阳性对照CCCP产生的蛋白变化相同。结果表明,喹烯酮处理下的HepG2细胞发生了线粒体自噬。对线粒体自噬受体的western blot检测显示,在喹烯酮作用下OPTN变化不显著,而TAX1BP1、FUNDC2表达量显著升高。但是,在CCCP的作用下OPTN显著升高,TAX1BP1显著降低,FUNDC2变化不明显。结果表明,喹烯酮作用下的HepG2细胞中线粒体自噬受体的变化趋势与典型的线粒体自噬有很大不同。进一步的试验运用parkin-Flag高表达质粒转染HepG2细胞。结果显示,喹烯酮和CCCP对parkin-Flag蛋白量都有显著抑制。转染质粒后HepG2在喹烯酮和CCCP处理下的线粒体膜蛋白和自噬相关蛋白的变化趋势都进一步加强。并且,喹烯酮和CCCP都引起了HepG2细胞线粒体膜电位降低。但是,CCCP使HepG2细胞的线粒体数量显著下降,而喹烯酮对线粒体数量的影响并不显著,parkin转染对结果影响也不明显。结果表明,虽然喹烯酮能使线粒体膜电位下降,但是对线粒体数量没有明显影响。综上所述,喹烯酮对线粒体的损伤作用虽然启动了HepG2细胞中的线粒体自噬,但是并没能造成线粒体数量的减少。从以上研究中可以得出结论,喹烯酮能引起HepG2细胞发生凋亡、自噬通量升高、以及线粒体自噬的起始。证明程序性细胞死亡在喹烯酮的毒性作用中发挥了重要作用。
[Abstract]:As a kind of new veterinary drug developed independently in China, it is widely used as feed additive in the production practice of animal husbandry and aquaculture. It has good antibacterial and growth promoting effect. In the 90s of last century, a series of conventional toxicological tests proved the safety of the addition agent of the feed. But after entering the new century, food safety The importance of the whole is becoming more and more concerned. Quinolone belongs to quinoline compounds. In the same class, quinolone, Kaba oxygen, and quinolone and Kaba oxygen have been proved to have significant toxicity. The safety of quinolone, which has the same core structure, has also been reconsidered. The cell withering caused by quinolone recently in our laboratory. This study intends to further explore the molecular mechanism of cell death under the action of programmed cell death. This study is to further explore the molecular mechanism of cell death under the action of the cell programmed cell death. The results show that when combined with ML-7, the toxicity of HepG2 cells increases and the rate of cell apoptosis is further increased. The effect of mitochondrial membrane potential was enhanced and the exogenous and endogenous apoptotic pathways were more activated. The above results showed that the process of cell death induced by apoptosis could be enhanced by ML-7. Further studies showed that ML-7 stimulated the MAPKs signaling pathway caused by the transports and inhibited the Akt signal caused by the ML-7. And, after the use of MAPKs inhibitors and Akt agonists, the results showed that both Akt collaterals and the MAPKs pathway were involved in the apoptosis induced by the Akt. To sum up, the mechanism of the apoptosis was mediated by the Akt pathway and the MAPKs pathway. The specific mechanism was that the apoptosis was induced by p38, while ERK, JNK and Akt pathways In this process, apoptosis is inhibited. Autophagy is also known as programmed cell type II death. GFP-LC3 plasmid transfected with HepG2 cells treated with OCT found that the LC3 aggregation point increased in the cells. The conversion rate of LC3 was dependent on the concentration and time dependence of the HepG2. The results showed that the HepG2 cells were autophagy. Then, it was found that the expression of endoplasmic reticulum stress protein BiP and CHOP showed a time dependence and concentration dependence of CHOP, and the transcriptional activity of sec24D, HerpUD, and BiP increased in the concentration dependence of the concentration of the endoplasmic reticulum. The results showed that the endoplasmic reticulum in the HepG2 cells was induced. The cascade of 6/DAPK1/MRLC enhanced the transfer of mAtg9a, thereby promoting the formation of autophagic bodies. The conversion rate of autophagy LC3 was reduced after interfering with ATF6 and DAPK1. In summary, the induction of autophagic flux was induced by endoplasmic reticulum stress. The results of this study showed that the core regulatory proteins parkin and PINK1 of mitochondrial autophagy were both concentration dependent and time-dependent in the HepG2 cells acting on the function of the PINK1; the expression of mitochondrial membrane protein COX IV protein was reduced, the protein expression of Tom20 and Tim23 increased, and the protein produced by the positive CCCP was the same. Western blot detection of autophagic receptors in HepG2 cells showed that OPTN changes were not significant under the action of quinolone, and the expression of TAX1BP1 and FUNDC2 increased significantly. However, the OPTN increased significantly under the action of CCCP, TAX1BP1 decreased significantly, and FUNDC2 changes were not obvious. The results showed that the FUNDC2 changes were not obvious. The results showed quinene The change trend of mitochondrial autophagy receptor in HepG2 cells was very different from that of typical mitochondrial autophagy. Further experiments used parkin-Flag high expression plasmid to transfect HepG2 cells. The results showed that the amount of parkin-Flag protein was significantly inhibited by the parkin-Flag and the transfected plasmid. The transfected plasmid was treated with the treatment of ketonone and CCCP. The change trend of mitochondrial membrane protein and autophagy related protein was further strengthened. Furthermore, the mitochondrial membrane potential decreased in HepG2 cells. However, CCCP decreased the number of mitochondria in HepG2 cells, and the effect of parkin on the number of mitochondria was not obvious, and the effect of parkin transfection on the results was not obvious. It was shown that although the membrane potential of the mitochondrial membrane decreased, but it had no obvious effect on the number of mitochondria. In summary, the damage of the mitochondria to mitochondria started the mitochondrial autophagy in HepG2 cells, but it did not cause a decrease in the number of mitochondria. Apoptosis, increased autophagy and initiation of mitochondrial autophagy have demonstrated that programmed cell death plays an important role in the toxicity of the compound.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S859.8

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