双氯芬酸对烟草BY-2细胞呼吸代谢及其线粒体功能的影响

发布时间：2019-03-28 20:52

【摘要】：随着经济的迅速发展,人类的医疗卫生条件不断改善,多达3000种药物被用于医疗,并在环境中积累。由于污水处理厂的设备并不能将这些有机药物完全去除,因此这些有机药物被排放到水环境中,不断渗透到地表水、地下水和沉积物中,使其在环境中的分布具有全球普遍性。其中,双氯芬酸是一种最常用的、疗效最好的非甾类镇痛消炎药(NSAIDs),被广泛用于临床治疗中。由于双氯芬酸药品的疗效好、用量大,被无节制地排放到环境中,并危及到了生态系统的稳定性。现在我们已知,双氯芬酸能严重影响植物的生长发育,但是其抑制植物生长的作用机制至今尚不清楚。为此,本研究利用烟草BY-2细胞为材料,探讨双氯芬酸对烟草BY-2细胞的呼吸代谢及其线粒体功能的影响,利用Oxytherm氧电极(Hansatech,英国)测定不同浓度的双氯芬酸钠即时处理的烟草BY-2细胞的总呼吸,测定不同处理时间的烟草BY-2细胞的总呼吸、细胞色素途径(COX)和交替氧化酶(AOX)途径呼吸以及线粒体的状态呼吸,测定不同呼吸途径抑制剂处理的细胞呼吸,并利用称量细胞干重的方法更直观的验证双氯芬酸钠对烟草BY-2细胞生长的抑制。进而,阐明双氯芬酸抑制烟草BY-2细胞生长的作用机理,为阐明双氯芬酸抑制植物生长的机理提供理论依据。本研究结果表明,0.2 mmol·L-1双氯芬酸处理24h后,就显著抑制了烟草BY-2细胞的生长,引起细胞内活性氧(ROS)的爆发和积累,并导致烟草BY-2细胞死亡。并且,研究发现一定浓度的双氯芬酸对烟草BY-2细胞的呼吸有即时的抑制作用,且随着双氯芬酸的浓度增加,对细胞呼吸作用的抑制程度也增加,当双氯芬酸的浓度达到0.2mmol·L-1时,它对烟草BY-2细胞呼吸作用的抑制程度达到最大。此外,在双氯芬酸对烟草BY-2细胞离体线粒体的直接作用的研究中,我们发现双氯芬酸通过抑制细胞线粒体呼吸电子传递链上复合体II、III和IV的活性,导致线粒体中细胞色素氧化酶(COX)和交替氧化酶(AOX)参与的两条呼吸电子传递途径均受到不同程度的抑制作用,进而反馈抑制了细胞的糖酵解(EMP)、三羧酸循环(TCA)和戊糖磷酸途径(PPP)三个碳代谢途径。并且,双氯芬酸对线粒体I、III、IV三个状态的呼吸均有抑制作用,导致呼吸控制率(RCR)降低。而且,双氯芬酸也能导致线粒体发生肿胀,并能降低线粒体膜磷脂的聚集程度,破坏了线粒体的完整性,导致跨膜质子梯度形成受阻,进而造成ATP合成受抑。因此,我们认为烟草BY-2细胞中经过线粒体呼吸电子传递链上复合体III和IV的电子传递被抑制是双氯芬酸抑制细胞呼吸的一个主要原因。本研究证明了双氯芬酸对线粒体呼吸链上复合体II,III和IV活性的抑制是它抑制细胞呼吸的主要原因,这就导致了线粒体代谢发生紊乱。线粒体能量代谢和物质代谢的紊乱进一步造成细胞内活性氧(ROS)爆发和积累,这些都是双氯芬酸抑制烟草BY-2细胞生长、导致细胞死亡的重要原因。
[Abstract]:With the rapid development of the economy, human health conditions have been improving, as many as 3,000 drugs have been used for medical treatment and are accumulated in the environment. Since the equipment of the sewage treatment plant cannot completely remove these organic drugs, these organic drugs are discharged into the water environment and continuously penetrate into the surface water, the ground water and the sediment, so that the distribution of the organic medicine in the environment is universal. Among them, diclofenac is one of the most commonly used non-opioid analgesic and anti-inflammatory drugs (NSAIDs), which are widely used in clinical treatment. Due to the good curative effect of the diclofenac medicine, the dosage is large, and the diclofenac medicine is discharged to the environment without control, and the stability of the ecological system is endangered. It is now known that diclofenac can severely affect the growth and development of plants, but the mechanism to inhibit plant growth is not yet clear. To this end, the effects of diclofenac on the respiratory metabolism and the mitochondrial function of the tobacco by-2 cells were investigated by using the tobacco BY-2 cells, and the total respiration of the tobacco by-2 cells treated with diclofenac sodium at different concentrations was determined by using the Oxytherm oxygen electrode (Hansatch, UK). the total respiration, the cytochrome pathway (COX) and the alternating oxidase (AOX) pathway of the tobacco by-2 cells and the state respiration of the mitochondria were measured for different treatment times, and the cellular respiration of the different respiratory pathway inhibitor treatment was determined, And the inhibition of the growth of the tobacco by-2 cells by the diclofenac sodium is more visually verified by the method for weighing the dry weight of the cells. In addition, the mechanism of diclofenac to inhibit the growth of the tobacco by-2 cells is explained, and the theoretical basis for elucidating the mechanism of diclofenac to inhibit the growth of the plants is provided. The results of this study show that after 24 h of 0.2 mmol 路 L-1 diclofenac acid treatment, the growth of the tobacco by-2 cells is significantly inhibited, and the occurrence and accumulation of reactive oxygen (ROS) in the cells is induced, and the tobacco by-2 cells are caused to die. In addition, it was found that the concentration of diclofenac has an immediate effect on the respiration of the tobacco by-2 cells, and as the concentration of diclofenac increases, the degree of inhibition of cellular respiration is also increased, and when the concentration of the diclofenac is 0.2 mmol 路 L-1, It has the greatest inhibitory effect on the respiration of the tobacco by-2 cells. In addition, in the study of the direct effect of diclofenac on the in vitro mitochondria of the tobacco by-2 cells, we found that diclofenac has been used to inhibit the activity of the complexes II, III and IV on the mitochondrial respiratory electron transfer chain of the cell, The two respiratory electron transport pathways involved in the participation of the cytochrome oxidase (COX) and the alternating oxidase (AOX) in the mitochondria are inhibited by different degrees, and the glycolysis (EMP) of the cells is further suppressed, The three pathways of carbon metabolism in the three-acid cycle (TCA) and the pentose phosphate pathway (PPP). In addition, diclofenac has an inhibitory effect on the respiration of the three states of the mitochondria I, III and IV, leading to a reduction in the respiratory control rate (RCR). In addition, diclofenac can also lead to the swelling of the mitochondria, and the aggregation of the mitochondrial membrane phospholipids can be reduced, the integrity of the mitochondria is destroyed, the formation of the transmembrane proton gradient is blocked, and the ATP synthesis is inhibited. Therefore, we believe that the inhibition of the electron transfer of complexes III and IV through the mitochondrial respiratory electron transfer chain in the tobacco BY-2 cells is a major cause of diclofenac to inhibit cellular respiration. This study has shown that the inhibition of the activity of diclofenac on the complex II, III and IV of the mitochondrial respiratory chain is the main cause of the inhibition of cellular respiration, which leads to a disorder in the metabolism of the mitochondria. The disorder of mitochondrial energy metabolism and substance metabolism further causes the explosion and accumulation of reactive oxygen (ROS) in the cells, which are important reasons for diclofenac to inhibit the growth of the tobacco by-2 cells and lead to cell death.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X503.231

【共引文献】