沙丁胺醇对黑腹果蝇基因组稳定性、细胞凋亡和蛋白表达的影响

Effects of salbutamol on the genome stability, apoptosis and protein expression in Drosophila melanogaster

【目的】已有研究表明,食用了饲喂以沙丁胺醇为主要成分的瘦肉精的动物肉类后,瘦肉精成分会在人体内富集,摄入过量沙丁胺醇会对生物体造成不良影响,但是,其具体毒性作用机理目前尚不明确。作为一种模式生物,黑腹果蝇Drosophila melanogaster与哺乳动物的基因具有较高的同源性,且具有繁殖周期短、方便进行遗传操作等优势。因此,我们通过研究过量沙丁胺醇对黑腹果蝇基因组稳定性、细胞凋亡和蛋白表达的影响,来探究它对生物体毒性作用的机理。【方法】将野生型黑腹果蝇3龄幼虫用含沙丁胺醇(120μg/m L)的饲料饲喂2 h后,对幼虫翅成虫盘进行H2Av抗体免疫染色。选取rpr-lac Z转基因黑腹果蝇1龄幼虫用含沙丁胺醇(40和120μg/m L)的饲料饲喂,对幼虫翅成虫盘进行lac Z活性测定。提取沙丁胺醇处理后的野生型3龄幼虫总蛋白,采用SDS-PAGE比较对照组和实验组蛋白表达的差异,并通过质谱分析差异蛋白的氨基酸序列。【结果】沙丁胺醇处理后,经免疫荧光染色发现野生型黑腹果蝇幼虫翅成虫盘部分细胞中组蛋白H2Av的量有显著增加。随着沙丁胺醇浓度的增加,转rpr-lac Z报告基因黑腹果蝇成虫盘细胞lac Z活性增强。采用SDS-PAGE和质谱分析表明,沙丁胺醇处理后黑腹果蝇肌动蛋白(Actin-87E)和异柠檬酸脱氢酶表达量上升。【结论】沙丁胺醇处理会引起黑腹果蝇细胞核DNA损伤,对基因稳定性有显著影响,并且会促进细胞凋亡和蛋白表达的改变。沙丁胺醇可能通过促进肌肉收缩和加速生物体能量代谢这两方面来减少脂肪积蓄。

[ Aim ] Previous studies have shown that salbutamol, a main component of lean meat powder, can be accumulated in human body after ingestion of meat containing it. Excessive salbutamol intakes would lead to a series of adverse reaction in organisms, but its toxic action mechanism remains unclear. Genes of the model organism Drosophila melanogaster show high homology to those of mammals, and D. melanogaster has the advantages of short reproductive cycle, easy genetic operation and so on. To explore its toxic mechanism, we studied the effects of excessive salbutamol on genome stability, apoptosis and protein expression in D. melanogaster. [ Methods ] After the 3rd instar larvae of the wild type and transgenic rpr-lacZ strains of D. melanogaster were fed with the artificial diet containing 120 μg/mL salbutamon for 2 h, their imaginal discs were dissected for histone H2Av immnofluorescent staining and measurement of the lacZ activity, respectively. Total proteins the wild type strain treated with salbutamol and separated by staining results showed that histone H2Av in some imaginal melanogaster larvae increased significantly as compared with salbutamol concentration, the lacZ activity of imaginal cells was were extracted from the 3rd instar larvae of SDS-PAGE. [ Results ] Immnofluorescent discs cells of the salbutamol treated D. that in the control. With the increment of enhanced in the rpr-lacZ reporter transgenic strain. The SDS-PAGE and MS-MALDI-TOF/TOF results showed that the expression levels of actin and isocitrate dehydrogenase increased in the larvae treated with salbutamol. [ Conclusions ] Salbutamol treatment affects the stability of nuclear DNA, promotes the apoptosis and changes the protein expression in D. melanogaster. Salbutamol might increase actin expression to promote muscle contraction and accelerate energy metabolism.