邻苯二甲酸二(2-乙基)己酯对INS-1细胞氧化应激和凋亡的影响
Effects of di(2-ethylhexyl) phthalate on apoptosis and oxidatve stress of INS-1 cells
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摘要:
目的 邻苯二甲酸二（2-乙基）己酯（DEHP）是一种具有代表性的邻苯二甲酸酯类增塑剂，常被用于塑料制品的生产加工，暴露来源广泛。有流行病学证据表明DEHP暴露与糖尿病的发生发展有一定关联，但鲜见相关基础研究。本研究探究DEHP对大鼠胰岛β细胞（INS-1细胞）氧化应激及细胞凋亡的影响。
方法 体外培养INS-1细胞，并用30、60、120 μmol/L的DEHP处理24 h。用MTT法检测细胞增殖抑制率，流式细胞仪检测细胞凋亡率。用2'，7'-二氯荧光黄双乙酸盐（DCFH-DA）检测细胞内活性氧（ROS）含量，可溶性噻唑盐（WST-8）法检测细胞内超氧化物过氧歧化酶（SOD）活性，5，5'-二硫代双（2-硝基苯甲酸）（DTNB）比色法检测还原型谷胱甘肽（GSH）及氧化型谷胱甘肽（GSSG）含量，硫代巴比妥酸（TBA）法检测脂质过氧化产物丙二醛（MDA）含量，用JC-1检测线粒体膜电位。用60、120、240 μmol/L的DEHP作用于INS-1细胞24 h后，用Westernblot检测凋亡相关蛋白Bax、Bcl-2、细胞色素C（Cyt-C）、Caspase-3、Caspase-9的表达。
结果 MTT实验和细胞凋亡检测结果显示，DEHP可导致INS-1细胞的增殖抑制，且120μmol/LDEHP还会引起凋亡（P < 0.05）。与对照组相比，30、60、120 μmol/L的DEHP可造成INS-1细胞内ROS荧光强度分别增强13.9%、20.7%和33.7%，MDA含量分别增加42.9%、44.8%和67.7%（P < 0.01）；60、120μmol/L的DEHP可造成细胞内GSH含量分别下降9.2%和54.8%，GSSG含量分别增加101.2%和444.1%，且还原型与氧化型谷胱甘肽的比值GSH/GSSG分别下降54.8%和91.7%（P < 0.05）；120 μmol/L的DEHP可造成SOD活性降低26.4%（P < 0.05）；30、60、120 μmol/L的DEHP可导致INS-1细胞线粒体膜电位降低，由此引起红绿荧光比值分别降低10.6%、36.8%和38.4%（P < 0.01）。Western blot结果显示，与对照组相比，60、120、240μmol/L的DEHP染毒组中，Cyt-C相对表达量增加；120、240μmol/L DEHP组Bax/Bcl-2值升高；240μmol/LDEHP组Caspase-9和Capase-3相对表达量也增加（P < 0.05）。
结论 DEHP可引起INS-1细胞氧化应激，降低线粒体膜电位，激活细胞凋亡线粒体通路相关蛋白的表达，诱导细胞凋亡。
Abstract:Objective Di(2-ethylhexyl) phthalate (DEHP), a representative plasticizer, is commonly used in plastcs producton and widely present in environment. Epidemiological studies have proved that DEHP is associated with the occurrence and development of diabetes, but the mechanism research is exiguous. The current study is conducted to explore the oxidatve stress and apoptosis in rat islet β cells (INS-1 cells) induced by DEHP.
Methods INS-1 cells were cultured in vitro, following exposure to 30, 60, and 120 μmol/L DEHP for 24 h. MTT assay was used to measure the cell proliferation inhibition rate, and flow cytometry for cell apoptosis. Cellular reactve oxygen species (ROS) generaton was detected by 2', 7'-dichlorodi-hydrofluorescein diacetate (DCFH-DA) method, the actvity of superoxide dismutase (SOD) by water soluble tetrazolium-8 (WST-8) method, reduced and oxidized glutathione (GSH and GSSG) by 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) method, malondialdehyde (MDA) by thiobarbituric acid (TBA) method, and mitochondrial membrane potential (MMP) by JC-1 staining. The cell apoptosis associated proteins, including Bax, Bcl-2, cytochrome C (Cyt-C), Caspase-3, and Caspase-9, were detected by Western blot afer exposure to 60, 120, and 240μmol/L DEHP for 24h.
Results The MTT and flow cytometry results showed that DEHP induced cell proliferation inhibition, and 120 μmol/L DEHP caused apoptosis (P < 0.05). Compared with the control group, the cellular ROS fluorescence intensites in the 30, 60, and 120 μmol/L DEHPexposed cells increased by 13.9%, 20.7%, and 33.7%, respectvely, and the MDA increased by 42.9%, 44.8%, and 67.7%, respectvely (P < 0.01); the cellular GSH in the 60 and 120 μmol/L DEHP-exposed cells decreased by 9.2% and 54.8%, the GSSG increased by 101.2% and 444.1%, and the GSH/GSSG decreased by 54.8% and 91.7%, respectvely (P < 0.05); the cellular SOD actvity in the 120 μmol/L DEHPexposed cells decreased by 26.4% (P < 0.05); the MMP red-to-green fluorescence ratos in the 30, 60, and 120 μmol/L DEHP-exposed cells decreased by 10.6%, 36.8% and 38.4%, respectvely (P < 0.01). The results of Western blot showed that compared with the control group, 60, 120, and 240 μmol/L DEHP treatments increased Cyt-C expression levels; 120 and 240 μmol/L DEHP treatments increased Bax/Bcl-2; 240μmol/L DEHP treatment increased Caspase-3 and Caspase-9 expression levels (P < 0.05).
Conclusion DEHP may promote oxidatve stress, reduce MMP, and induce cell apoptosis by actvatng proteins related to mitochondrial pathway.
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