糖尿病肾病细胞模型三羧酸循环代谢异常和机制初探

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。qPCR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。

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

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 fluorescence microscopy after mitochondrial staining. Metabolic disruption was evaluated by a GC-MS based metabolomics method. Concentration of TCA intermediates was measured by a GC-MS based quantification method. Expressions of relative enzymes were measured by qPCR method. RESULTS:High glucose did not exert as strong an effect on cell damage or metabolic disruption as PA, which caused obvious cell damage or metabolic disruption in a short period. The combined inducement presented the strongest effect. TCA intermediates variation was the main change in PA caused HK-2 cells metabolic disruption. 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. CONCLUSION: Metabolic disruption of TCA induced by PA may be closely related to diabetic nephropathy.