李丽萍, 王乐, 德小明, 李玲, 张亚娟, 朱丽敏. 双酚A对大鼠胰岛素瘤细胞凋亡及胰岛素分泌水平的影响[J]. 环境与职业医学, 2020, 37(6): 609-615. DOI: 10.13213/j.cnki.jeom.2020.19793
引用本文: 李丽萍, 王乐, 德小明, 李玲, 张亚娟, 朱丽敏. 双酚A对大鼠胰岛素瘤细胞凋亡及胰岛素分泌水平的影响[J]. 环境与职业医学, 2020, 37(6): 609-615. DOI: 10.13213/j.cnki.jeom.2020.19793
LI Li-ping, WANG Le, DE Xiao-ming, LI Ling, ZHANG Ya-juan, ZHU Li-min. Effects of bisphenol A on apoptosis and insulin secretion of insulinoma cells in rats[J]. Journal of Environmental and Occupational Medicine, 2020, 37(6): 609-615. DOI: 10.13213/j.cnki.jeom.2020.19793
Citation: LI Li-ping, WANG Le, DE Xiao-ming, LI Ling, ZHANG Ya-juan, ZHU Li-min. Effects of bisphenol A on apoptosis and insulin secretion of insulinoma cells in rats[J]. Journal of Environmental and Occupational Medicine, 2020, 37(6): 609-615. DOI: 10.13213/j.cnki.jeom.2020.19793

双酚A对大鼠胰岛素瘤细胞凋亡及胰岛素分泌水平的影响

Effects of bisphenol A on apoptosis and insulin secretion of insulinoma cells in rats

  • 摘要: 背景

    双酚A(BPA)是一种典型的具有拟雌激素作用的环境内分泌干扰物。越来越多的研究显示,BPA暴露与人类2型糖尿病(T2DM)发生呈正相关,但其作用机制尚不清楚。探讨BPA对糖代谢的影响具有非常重要的意义。

    目的

    探讨双酚A对大鼠胰岛素瘤细胞(INS-1细胞)凋亡及胰岛素分泌水平的影响。

    方法

    常规培养INS-1细胞,待细胞贴壁后,用含不同浓度BPA(6、30、150 μmol·L-1)的RPMI 1640培养基进行染毒,以DMSO为溶剂(终浓度≤ 0.1%)。采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测BPA对INS-1细胞存活率的影响;流式细胞术检测BPA对INS-1细胞凋亡率的影响;活性氧(ROS)试剂盒检测BPA染毒对INS-1细胞ROS水平的影响;葡萄糖刺激胰岛素分泌实验检测不同浓度BPA对INS-1细胞胰岛素分泌的影响;荧光定量聚合酶链反应、蛋白质印迹法分别检测BPA对INS-1细胞Bcl-2、Bax、半胱天冬酶(caspase)3基因及其蛋白表达水平的影响。

    结果

    与DMSO溶剂对照组比较,30 μmol·L-1 BPA组INS-1细胞存活率增高,150 μmol·L-1 BPA组细胞存活率降低(P < 0.01)。流式细胞术检测结果显示:6、150μmol·L-1 BPA组INS-1细胞总凋亡率增高(均P < 0.05)。6、30、150 μmol·L-1 BPA组INS-1细胞内ROS含量增加(均P < 0.05)。葡萄糖刺激胰岛素分泌实验结果显示:在基础浓度葡萄糖(5.6 mmol·L-1)刺激下,6、30 μmol·L-1 BPA组INS-1细胞胰岛素分泌水平增高(均P < 0.05),150 μmol·L-1 BPA组胰岛素分泌水平下降(P < 0.01),而在高浓度葡萄糖(16.7 mmol·L-1)刺激下,30 μmol·L-1 BPA组胰岛素分泌水平增高(P < 0.05),6、150 μmol·L-1 BPA组胰岛素分泌水平下降(均P < 0.05)。抑制凋亡基因Bcl-2的mRNA及蛋白相对表达水平在6、150 μmol·L-1 BPA组降低,在30 μmol·L-1 BPA组增高(均P < 0.05);促进凋亡基因Bax的mRNA和蛋白相对表达水平在150 μmol·L-1 BPA组增高(均P < 0.05);caspase3的mRNA和蛋白相对表达水平在150 μmol·L-1BPA组增高,在30 μmol·L-1BPA组降低(均P < 0.05);Bcl-2Bax的mRNA和蛋白相对表达比值在6、150 μmol·L-1 BPA组降低(均P < 0.05);cleaved-caspase3与caspase3蛋白相对表达水平比值在6、150 μmol·L-1 BPA组增高(均P < 0.05)。

    结论

    30 μmol·L-1BPA对INS-1细胞具有一定的促进增殖作用,对INS-1细胞胰岛素分泌水平也具有促进作用;6、150 μmol·L-1BPA可降低INS-1细胞活力,诱导INS-1细胞凋亡,降低胰岛素分泌水平,其作用机制可能与氧化损伤有关。

     

    Abstract: Objective

    This experiment investigates the effects of BPA on apoptosis and insulin secretion of insulinoma cells (INS-1 cells) in rats.

    Methods

    INS-1 cells were routinely cultured and exposed in RPMI 1640 medium containing different concentrations of BPA (6, 30, and 150 μmol·L-1, respectively), with DMSO as solvent (final concentration ≤ 0.1%). Methyl thiazolyl tetrazolium assay was used to detect survival rate of INS-1 cells; flow cytometry was used to detect apoptosis rate of INS-1 cells; reactive oxygen species (ROS) kit was used to detect ROS level of INS-1 cells; glucose-stimulated insulin secretion assay was used to detect insulin secretion of INS-1 cells; reverse transcription polymerase chain reaction and Western blotting were used to detect expressions of Bcl-2, Bax, caspase3 genes and their proteins in INS-1 cells, respectively.

    Results

    Compared with the DMSO solvent control group, the survival rate of INS-1 cells was increased in the 30 μmol·L-1 BPA group and decreased in the 150 μmol·L-1 BPA group (P < 0.01). The results of flow cytometry showed that the total apoptosis rate of INS-1 cells was increased in the 6 and 150 μmol·L-1 BPA groups (P < 0.05). The content of ROS in INS-1 cells was increased in the 6, 30, and 150 μmol·L-1 BPA groups (P < 0.05). The results of glucose-stimulated insulin secretion assay showed that under the stimulation of basic concentration of glucose (5.6 mmol·L-1), the level of insulin secretion of INS-1 cells was increased in the 6 and 30 μmol·L-1 BPA groups, and decreased in the 150 μmol·L-1 BPA group (P < 0.05); under the stimulation of high concentration of glucose (16.7 mmol·L-1), the level of insulin secretion was increased in the 30 μmol·L-1 BPA group (P < 0.05), and decreased in the 6 and 150 μmol·L-1 BPA groups (P < 0.05). The mRNA and protein relative expression levels of anti-apoptotic gene Bcl-2 were decreased in the 6 and 150 μmol·L-1 BPA groups and increased in the 30 μmol·L-1 BPA group (P < 0.05); the mRNA and protein relative expression levels of pro-apoptotic gene Bax were increased in the 150 μmol·L-1 BPA group (P < 0.05); the mRNA and protein relative expression levels of caspase3 were increased in the 150 μmol·L-1 BPA group and decreased in the 30 μmol·L-1 BPA group (P < 0.05), the ratios of mRNA and protein relative expression levels of Bcl-2 to Bax were decreased in the 6 and 150 μmol·L-1 BPA groups; the ratio of protein relative expression level of cleaved-caspase3 to caspase3 was increased in the 6 and 150 μmol·L-1 BPA groups (P < 0.05).

    Conclusion

    BPA administration at 30μmol·L-1 could promote proliferation and insulin secretion of INS-1 cells, while that at 6 and 150 μmol·L-1 could reduce the viability of INS-1 cells, induce apoptosis, and decrease the level of insulin secretion, which may be related to oxidative damage.

     

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