糖尿病肾病细胞模型三羧酸循环代谢异常和机制初探  

Preliminary study on mechanism of tricarboxylic acid cycle disruption in diabetic nephropathy cell model

作　　者：俞晓忆[1] 周芷若[2] 谢媛[1] 孙润彬 费菲[1] 高浩雪 黄京秋 阿基业[1] 王广基[1] 

机构地区：[1]中国药科大学药科院药物代谢动力学重点实验室 [2]南京中医药大学药学院药效与安全性评价重点实验室

出　　处：《中国临床药理学与治疗学》2017年第5期481-489,共9页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：江苏省科技计划项目临床医学科技专项(BL2014070);国家自然科学基金项目(81072692);973"国家重点基础研究发展计划资助"子课题(2012CB517606)

摘　　要：目的:探讨糖尿病肾病(DN)状态下三羧酸(TCA)循环中间代谢产物的差异,观察糖尿病肾病细胞模型中的代谢紊乱。方法:体外培养人肾小管上皮细胞(HK-2),分为对照组(Control)、高糖组(High glucose)、棕榈酸组(PA)、棕榈酸复合高糖组(PA high glucose)。线粒体染色法观察造模后的细胞损伤状况,基于气相的代谢组学方法检测细胞模型中出现的代谢差异,并找出相关差异化合物。气相色谱-质谱(GC-MS)定量方法进一步测定差异化合物的绝对浓度。q PCR测定差异化合物相关通路的酶表达。结果:12、24 h内高糖对HK-2细胞的损伤和代谢影响较弱,而棕榈酸在短期内就可以造成肾脏细胞损伤和代谢紊乱,复合因素下的诱导效果更甚。造模细胞内出现的代谢异常中TCA循环占主要部分,其中柠檬酸和琥珀酸水平出现异常升高。并且琥珀酸水平异常主要由其合成酶影响导致。结论:棕榈酸诱导引起细胞中TCA循环代谢异常变化可能与DN相关。AIM： To explore the difference of tricarboxylic acid cycle （TCA） intermediates levels between diabetic nephropathy （DN） model cells and normal HK-2 cells and the metabolic disruption of DN model. METHODS： HK-2 cells were cultured in vitro and were divided into normal group, glucose group, palmitic acid （PA） group, PA high glucose group. Kidney cell damage was observed by fluores- cence microscopy after mitochondrial staining. Meta- bolic disruption was evaluated by a GC-MS based metabolomics method. Concentration of TCA inter- mediates was measured by a GC-MS based quantifi- cation method. Expressions of relative enzymes were measured by qPCR method. RESULTS： High glu- cose did not exert as strong an effect on cell damage or metabolic disruption as cell damage or metabolic PA, which caused obvious disruption in a short peri- od. The combined inducement presented the stron- gest effect. TCA intermediates variation was the main change in PA caused HK-2 cells metabolic dis- ruption. Further, PA could increase the level of citrate and succinate in HK-2 cells. And instead of SDH in model cell, the increase of succinate level was closely related to succinate-CoA ligase. CON- CLUSION： Metabolic disruption of TCA induced by PA may be closely related to diabetic nephropathy.

关 键 词：糖尿病肾病 人肾小管上皮细胞 棕榈酸 琥珀酸 

分 类 号：R965.2[医药卫生—药理学]