邻苯二甲酸二(2-乙基己基)酯通过氧化应激介导胰岛β细胞自噬通量受损对大鼠糖稳态的影响

Effects of di(2-ethylhexyl) phthalate on glucose homeostasis in rats due to impaired autophagy flux of islet β cells mediated by oxidative stress

  • 摘要:
    背景 邻苯二甲酸二(2-乙基己基)酯(DEHP)是全球用量最大的邻苯二甲酸盐类环境内分泌干扰物,研究表明DEHP暴露对机体糖代谢可能产生影响。
    目的 探讨DEHP通过氧化应激介导胰岛β细胞自噬通量受损对大鼠糖稳态影响的潜在机制。
    方法 40只SPF级雄性SD大鼠随机分为四组,DEHP(0、187、375、750 mg·kg−1)经口灌胃染毒12周,于末次染毒24 h后分别进行口服葡萄糖耐量试验(OGTT)和胰岛素耐量试验(ITT)。苏木精-伊红(HE)染色、透射电子显微镜(TEM)观察胰腺组织病理形态改变;酶联免疫吸附法检测大鼠血清胰岛素、三磷酸腺苷(ATP)、单磷酸腺苷(AMP)水平、胰腺组织活化的半胱天冬蛋白酶-3(cleaved Caspase-3)蛋白水平;微板法检测血清中还原性谷胱甘肽(GSH)含量、水溶性四唑盐-1法检测超氧化物歧化酶(SOD)含量、硫代巴比妥酸法检测丙二醛(MDA)含量;采用化学荧光法检测胰腺组织活性氧(ROS)含量;用逆转录聚合酶链反应(RT-PCR)法检测螯合体1(SQSTM1/p62)、自噬相关蛋白苄氯素1(Beclin1)、微管相关蛋白1A/1B-轻链3(LC3)、半胱天冬蛋白酶-8(Caspase-8)mRNA水平;Western blot法检测p62、Beclin-1、LC3-I、LC3-II单磷酸腺苷活化蛋白激酶(AMPK)、p-AMPK、哺乳动物雷帕霉素靶蛋白(mTOR)、p-mTOR、UNC-51样激酶1(ULK1)、p-ULK1、Caspase-8蛋白表达水平。
    结果 与0 mg·kg−1 DEHP组相比,750 mg·kg−1 DEHP组在第2、4、6、12周时空腹血糖均升高(P<0.05)。OGTT结果显示,高糖灌胃后,与0 mg·kg−1 DEHP组相比,187 mg·kg−1 DEHP组在30 min时血糖水平上升(P<0.05);375 mg·kg−1 DEHP组在15、30、180 min时血糖水平上升(P<0.05);750 mg·kg−1 DEHP组在15、30、60、180 min时血糖水平上升(P<0.05);375、750 mg·kg−1 DEHP组OGTT曲线下面积(AUC)增加(P<0.05)。ITT结果显示,经腹腔注射胰岛素后,与0 mg·kg−1 DEHP组相比,各DEHP染毒组间各时间点血糖水平、ITT AUC差异均无统计学意义(P>0.05)。与0 mg·kg−1 DEHP组相比,750 mg·kg−1 DEHP组大鼠HOMA-IR升高、HOMA-ISI降低(P<0.05)。HE和TEM结果显示:各DEHP染毒组中,胰岛细胞数量减少,胰岛面积减少,并伴随着染色质凝集,胰岛β细胞胞质内分泌颗粒减少,出现不同程度的核膜间隙增宽、高尔基复合体扁平毛囊扩张、内质网扩张,出现核糖体分离现象,可见自噬小体,375、750 mg·kg−1 DEHP组线粒体有不同程度的变形,部分嵴结构消失呈现空泡化,且750 mg·kg−1 DEHP组细胞核染色质凝集现象较为严重。750 mg·kg−1 DEHP组血清中SOD活力升高(P<0.05);375、750 mg·kg−1 DEHP组胰腺组织中ROS相对含量升高(P<0.05);各DEHP染毒组MDA含量均升高(P<0.05)、血清GSH含量均降低(P<0.05);750 mg·kg−1 DEHP组血清AMP/ATP的比值上升(P<0.05),大鼠胰腺组织cleaved Caspase-3蛋白表达水平上升(P<0.05)。各DEHP染毒组大鼠胰腺组织p62Beclin-1LC3Caspase-8 mRNA相对水平均升高(P<0.05);各DEHP染毒组p-AMPK/AMPK、p-ULK1/ULK1、Beclin-1蛋白相对表达水平升高(P<0.05);375、750 mg·kg−1 DEHP组p62、LC3-II/LC3-I、Caspase-8蛋白相对表达水平升高(P<0.05)、p-mTOR/mTOR相对表达水平降低(P<0.05)。
    结论 DEHP可引起大鼠糖稳态异常,其潜在机制可能是通过诱导氧化应激介导ROS/AMPK/ULK1信号通路激活胰岛β细胞自噬,进一步损害自噬通量进而诱导胰岛β细胞凋亡,引起胰岛β细胞功能和数量的降低,导致机体糖稳态异常。

     

    Abstract:
    Background Di(2-ethylhexyl) phthalate (DEHP) is the most prevalent environmental endocrine disruptor among phthalate acid esters (PAEs) worldwide. Previous studies have indicated that exposure to DEHP may disrupt glucose metabolism.
    Objective To investigate the impact of DEHP on glucose homeostasis in rats, focusing on oxidative stress-induced impairment of autophagy in islet β cells.
    Methods Forty male SD rats were randomly assigned to four groups, receiving DEHP doses of 0, 187, 375, and 750 mg·kg−1 for 12 weeks. Oral glucose tolerance (OGTT) and insulin tolerance tests (ITT) were conducted 24 h after the final exposure. Pancreatic microstructural alterations were assessed using hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM). Commercial ELISA kits were employed to quantify the levels of insulin, adenosine triphosphate (ATP), and adenosine monophosphate (AMP) in rat serum, as well as the protein expression level of activated caspase-3 in pancreatic tissue. Additionally, commercial microplate kits were utilized to measure the concentration of reduced glutathione (GSH) in serum, the activity of superoxide dismutase (SOD) using water-soluble tetrazolium salt-1, the content of malondialdehyde (MDA) by thiobarbituric acid method, and the level of reactive oxygen species (ROS) in pancreatic tissue by chemical fluorescence method. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure sequestosome1 (SQSTM1/p62), Beclin1, microtubule-associated protein 1 light chain 3 (LC3), and cysteinyl aspartate specific proteinase-8 (Caspase-8) mRNA levels. Western blot analysis was applied to detect the protein relative expression levels of p62, Beclin-1, LC3-I, LC3 II, AMPK, p-AMPK, mTOR, p-mTOR, ULK1, and Caspase-8.
    Results Compared to the 0 mg·kg−1 DEHP group, the 750 mg·kg−1 DEHP group exhibited a significant increase in fasting blood glucose levels at 2, 4, 6, and 12 weeks (P<0.05). The OGTT showed that, following high-glucose gavage, the 187 mg·kg−1 DEHP group had elevated blood glucose at 30 min (P<0.05), the 375 mg·kg−1 DEHP group showed increased glucose levels at 15, 30, and 180 min (P<0.05), and the 750 mg·kg−1 DEHP group exhibited elevated levels at 15, 30, 60, and 180 min (P<0.05). The 375 and 750 mg·kg−1 DEHP groups demonstrated significantly increased OGTT area under the curve (AUC) values (P<0.05). In contrast, ITT results indicated no significant differences in blood glucose levels or AUC among the DEHP exposure groups at all time points (P>0.05). Compared to the 0 mg·kg−1 DEHP group, the 750 mg·kg−1 DEHP group exhibited significantly higher HOMA-IR levels and markedly lower HOMA-ISI values (P<0.05). HE and TEM showed that in each DEHP exposure group, the number of islet cells decreased, the islet area reduced, and chromatin condensation occurred. The endocrine granules in the cytoplasm of islet β cells decreased, and there were varying degrees of widening of the nuclear membrane gap, flattening and expansion of the Golgi complex, and expansion of the endoplasmic reticulum. Ribosome separation was observed, and autophagosomes were visible. In the 375 and 750 mg·kg−1 DEHP groups, the mitochondria were deformed to varying degrees, and some cristae structures disappeared, presenting vacuolization. Moreover, the chromatin condensation in the nuclei was more severe in the 750 mg·kg−1 DEHP group. The serum SOD activity was significantly elevated in the 750 mg·kg−1 DEHP group (P<0.05). Both the 375 mg·kg−1 and 750 mg·kg−1 DEHP groups exhibited a significant increase in the relative ROS content in pancreatic tissue (P<0.05). In DEHP-treated groups, the MDA content increased (P<0.05), while the GSH content decreased (P<0.05). Additionally, in the 750 mg·kg−1 DEHP group, the AMP/ATP ratio in serum was significantly raised (P<0.05), and the expression of cleaved Caspase-3 protein in pancreatic tissue was also significantly increased (P<0.05). The relative mRNA levels of p62, Beclin-1, LC3, and Caspase-8 in the pancreatic tissue of rats exposed to DEHP were significantly elevated (P<0.05). The relative expression levels of p-AMPK/AMPK, p-ULK1/ULK1, and Beclin-1 proteins in the DEHP-treated groups were significantly increased (P<0.05). In the 375 mg·kg−1 and 750 mg·kg−1 DEHP treatment groups, the relative expression levels of p62, LC3 II/LC1, and Caspase-8 proteins were significantly increased (P<0.05), while the relative expression level of p-mTOR/mTOR was significantly decreased (P<0.05).
    Conclusion DEHP can disrupt glucose homeostasis by inducing oxidative stress, which subsequently activates autophagy via the ROS/AMPK/ULK1 pathway, impairing autophagic flux and promoting apoptosis of islet β cells, ultimately decreasing their function and number.

     

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