体外评价口服降糖药对HepG2细胞线粒体功能的影响
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国家“十二五”重大专项基金(2012ZX09302002,2012ZX09505-001-003);上海市研发平台与环境条件基地建设专项基金(14140900901)


In vitro assessment of mitochondrial dysfunction of hypoglycemic drug on HepG2 cells
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    目的:评价DPP- Ⅳ抑制剂类口服降糖药A化合物的线粒体毒性,从而探讨A化合物的可能毒性机制。方法:HepG2细胞培养基中分别加入曲格列酮50~300 μmol/L,培养24 h,或加入A化合物溶液100~300 μmol/L,培养24 h,CCK8细胞计数法测定细胞存活率。HepG2细胞培养基中分别加入曲格列酮100-200和225 μmol/L培养24 h,或加入A化合物溶液100-150和200 μmol/L培养24 h,化学发光法检测胞内ATP合成水平,荧光探针法检测胞内钙离子浓度-活性氧-线粒体膜电位(-驻Ψm)变化,透射电镜观察线粒体超微结构。结果:与溶媒对照组相比,曲格列酮50~300 μmol/L可以抑制细胞存活率,IC50为178 μmol/L;A化合物 100~300 μmol/L对细胞存活率有抑制作用,IC50为159 μmol/L。与溶媒对照组相比,曲格列酮≥200 μmol/L时导致线粒体ATP显著下降(P<0.01)--驻Ψm显著性下降(P < 0.01),≥100 μmol/L时可致活性氧水平和钙离子浓度显著升高(P < 0.05或P < 0.01)。A化合物≥100 μmol/L时ATP合成水平显著降低-钙离子浓度显著升高(P < 0.01),≥150 μmol/L时活性氧水平明显升高(P < 0.01),≥200 μmol/L时-驻Ψm显著性下降(P < 0.05),并可导致线粒体结构发生病变。结论:DPP- Ⅳ抑制剂类口服降糖药A化合物可以通过干扰线粒体代谢功能和结构而诱导线粒体损伤。

    Abstract:

    Objective:To evaluate the potential mitochondrial toxicity of A compound,one of DPP-Ⅳ inhibitor hypoglycemic drugs, and to explore the mechanisms of toxicity of A compound. Methods: HepG2 cells were cultured in troglitazone(TRO)50~300 μmol/L for 24 h or A compound 100~300 μmol/L for 24 h. The cell viability was assessed by CCK8 assay. HepG2 cells were cultured in TRO 100,200 and 225 μmol/L for 24 h or A compound 100,150 and 200 μmol/L for 24 h. The level of cellular ATP was detected by luciferase assay. The levels of intracellular free Ca2+,reactive oxygen species(ROS),mitochondrial membrane potential(-驻Ψm)and mitochondrial permeablity transition pore(MPTP)were determined using flow cytometer. Results: Compared with the vehicle control group,TRO at 50~300 μmol/L markedly inhibited cell viability(IC50=178 μmol/L),A compound at 100~300 μmol/L markedly inhibited cell viability(IC50=159 μmol/L). Compared with the vehicle control,levels of ATP and -驻Ψm were significantly decreased in the TRO treated group(≥200 μmol/L)(P < 0.01),levels of ROS were significantly decreased and intracellular free Ca2+ were significantly increased in the TRO treated group (≥100 μmol/L)(P < 0.05 or P < 0.01). After A compound treatment,compared with the vehicle control,levels of ATP and intracellular free Ca2+ were significantly decreased at 100 μmol/L(P < 0.01). A compound at 150 μmol/L increased ROS level significantly (P < 0.01),A compound at 200 μmol/L decreased -驻Ψm level significantly(P<0.05)and mitochondrial ultra-structural changes were observed. Conclusion: DPP-Ⅳ inhibitor A compound can induce mitochondrial toxicity by interfering with mitochondrial metabolism.

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富 欣,潘晓靓,李 华,汤纳平,王 雁,惠涛涛,张泽安.体外评价口服降糖药对HepG2细胞线粒体功能的影响[J].南京医科大学学报(自然科学版),2015,(5):644-649

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  • 收稿日期:2015-01-23
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  • 在线发布日期: 2015-05-22
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