牛磺酸保护三氧化二砷引起的子代大鼠胰腺自噬性损伤的实验研究

发布时间：2018-04-04 19:16

本文选题：牛磺酸　切入点：As_2O_3　出处：《大连医科大学》2017年硕士论文

【摘要】：目的:砷是存在于自然界中的环境污染物之一。无机砷的暴露会引起很多慢性疾病的发生,比如糖尿病。而母体的高砷暴露对子代的影响更是引起了越来越多的关注。自噬是通过清除失调线粒体更新线粒体生物源,通过线粒体伴侣分子及线粒体水解酶管理线粒体蛋白质量,从而达到健康线粒体数量的平衡的过程,然而自噬的失调会造成组织损伤,引起相关疾病。有研究表明,活性氧(ROS)的产生与砷引起的胰岛素抵抗及糖尿病有很大关系。而转录因子NF-E2相关因子(Nrf2)是细胞氧化应激反应中的关键因子,具有抗氧化作用,与机体的防御系统密切相关。本课题组已有研究证明砷可以引起大鼠肝脏的自噬性死亡,但其机制是否与Nrf2和Trx有关还需进一步研究。牛磺酸是天然存在于自然界中的抗氧化物质,但是否可以缓解砷诱导的胰腺损伤及其机制还不清楚。因此,本研究拟探讨牛磺酸可否通过Nrf2-Trx通路干预砷引起的子代大鼠胰腺的自噬性损伤。方法:本实验以Wistar大鼠为研究对象,孕鼠暴露于浓度为2 mg/kg BW~8mg/kg BW的三氧化二砷(As_2O_3),及150 mg/kg BW浓度牛磺酸灌胃后,进行8mg/kg BW As_2O_3灌胃,至子代断乳,后将子代大鼠继续暴露于与母鼠相同剂量的As_2O_3至性成熟,共57天。对子代大鼠胰腺进行苏木精-伊红(HE)染色,观察子代大鼠胰腺的形态;用电子显微镜观察子代大鼠胰腺细胞中自噬体的形成情况,计数;用蛋白质印迹法(Western blot)检测子代大鼠胰腺中Nrf2蛋白及自噬生物标记物LC3蛋白的表达情况;用RT-PCR方法检测子代大鼠胰腺中Trx基因的表达情况;利用MDA试剂盒测量胰腺组织中MDA的水平。结果:HE染色结果显示,随着As_2O_3浓度的增加,子代大鼠胰腺细胞及胰岛明显缩小,而在牛磺酸保护组,子代大鼠胰腺细胞形态未见明显异常;电子显微镜下可见,As_2O_3暴露后,子代大鼠胰腺中有自噬体形成,其数量随着As_2O_3浓度的升高而增多,牛磺酸保护组自噬体增多不明显;子代大鼠胰腺中LC3-II蛋白表达随着As_2O_3浓度的升高而增加,牛磺酸保护组LC3-II蛋白表达较8mg/kg BW As_2O_3暴露组LC3-II蛋白的表达量减少;子代大鼠胰腺中Nrf2蛋白,Trx基因及MDA的水平随着As_2O_3暴露剂量的升高而明显减少,而在牛磺酸保护组,其表达量较8 mg/kg BW As_2O_3暴露组蛋白表达量明显上升。结论:As_2O_3可引起的子代大鼠胰腺自噬性损伤;Nrf2-Trx的抑制造成As_2O_3引起的子代大鼠胰腺自噬性损伤;牛磺酸可通过激活Nrf2-Trx通路干预As_2O_3引起的子代大鼠胰腺自噬性损伤。
[Abstract]:Objective: arsenic is one of the environmental pollutants in nature.Exposure to inorganic arsenic can cause many chronic diseases, such as diabetes.More and more attention has been paid to the effects of high arsenic exposure on offspring.Autophagy is the process of renewing mitochondrial biological sources by clearing out dysfunctional mitochondria and managing mitochondrial protein content through mitochondrial chaperones and mitochondrial hydrolases, thus achieving a healthy mitochondrial balance.However, autophagy disorders can cause tissue damage, causing related diseases.Studies have shown that reactive oxygen species (Ros) production is associated with arsenic-induced insulin resistance and diabetes.The transcription factor NF-E2 related factor Nrf2 is a key factor in the oxidative stress response of cells. It has antioxidant effect and is closely related to the body's defense system.Our research has shown that arsenic can induce autophagic death in rat liver, but whether the mechanism is related to Nrf2 and Trx needs further study.Taurine is a natural antioxidant in nature, but it is not clear whether it can alleviate arsenic induced pancreatic injury and its mechanism.Therefore, the aim of this study was to investigate whether taurine could interfere with arsenic induced autophagy injury in rat pancreas via Nrf2-Trx pathway.Methods: in this experiment, Wistar rats were exposed to 2 mg/kg BW~8mg/kg BW arsenic trioxide (as _ 2O _ 2O _ 3) and 150 mg/kg BW taurine. After the rats were fed with 8mg/kg BW As_2O_3, the rats were fed with 8mg/kg BW As_2O_3 and then weaned in their offspring.Then the offspring rats were exposed to the same dose of As_2O_3 as the mother rat until sexual maturation for 57 days.The morphology of rat pancreas was observed by hematoxylin-eosin-hehe staining and the formation and count of autophagy in pancreatic cells of offspring rats were observed by electron microscope.The expression of Nrf2 protein and autophagy biomarker LC3 protein in rat pancreas was detected by Western blotting, and the expression of Trx gene was detected by RT-PCR method.The level of MDA in pancreatic tissue was measured by MDA kit.Results with the increase of As_2O_3 concentration, the pancreatic cells and islets of the offspring decreased significantly, but in the taurine protected group, the morphology of pancreatic cells in the offspring was not significantly abnormal, and that after the exposure of as _ 2O _ 3 was observed under the electron microscope.Autophagy was formed in the pancreas of offspring rats, and the number of autophagy increased with the increase of As_2O_3 concentration, but the increase of autophagy in taurine protected group was not obvious, and the expression of LC3-II protein in pancreas of offspring rats increased with the increase of As_2O_3 concentration.The expression of LC3-II protein in taurine protected group was lower than that in 8mg/kg BW As_2O_3 group, and the level of Nrf2 protein Trx gene and MDA in the pancreas of offspring decreased with the increase of As_2O_3 exposure dose, but in taurine protective group.The expression of histone was significantly higher than that of 8 mg/kg BW As_2O_3.Conclusion the inhibition of Nrf2-Trx in the pancreatic autophagy of offspring rats induced by% As2O3 can result in autophagy injury induced by As_2O_3, and taurine can interfere with the pancreatic autophagy injury induced by As_2O_3 by activating the Nrf2-Trx pathway.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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