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

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.