ALA/LA脂肪酸氧化对高糖损伤细胞和糖尿病肾病小鼠的作用及机制研究

Fatty Acid Oxidation (FAO) is the preferred energy source for highly metabolic cells such as tubular epithelial cells. Mitochondria metabolism regulated by FAO has a key role in epithelial-to-mesenchymal transition (EMT) and kidney fibrosis development. Alteration in cellular metabolism, will emerged as an important therapeutic approach for kidney diseases by targeting FAO. But very little is known about the effect and metabolism of essential fatty acid (alpha-linolenic acid，ALA/linoleic acid，LA) on it. Our previous results showed that ALA/LA improved recovery from the injury of HK-2 induced by high glucose and inhabited cell apoptosis. So we use HK-2 cell and diabetics kidney disease (DKD) db/db mice to expose the mechanism of ALA/LA with proper concentration and ratio improve FAO and mitochondria metabolism synthesis in HK-2 cell, reduce cell apoptosis, and slow the kidney fibrosis development via TGF-β1/PPAR-α/PCG-1α signal pathway. Methods including real-time PCR, Western Blot and siRNA are used in the experiment to observe the effect from multi-levels of molecule, cell, tissue and animal entirety. The study provides new sight on the treatments of DKD. It is meaningful to study on the function of essential fatty acids and application on DKD.

目前国外研究发现，脂肪酸氧化（FAO）产生的能量，是高能需要肾小管上皮细胞的主要来源， FAO调节线粒体能量代谢，是上皮细胞向间质细胞转化和肾脏纤维化进程的重要因素，以FAO为靶点调控上皮细胞能量代谢，可提供治疗相关疾病基础策略。但是必需脂肪酸ALA/LA对上皮细胞向间质细胞转化的作用及其机制，国内外尚缺乏研究。申请人根据自己前期工作发现，ALA/LA能够减轻高糖对HK-2细胞的损伤。故拟采用HK-2细胞和糖尿病肾病db/db小鼠，从分子、细胞、组织及整体动物水平等多方面,探讨适宜剂量和浓度的ALA/LA，经TGF-β1/PPAR-α/PCG-1α通路，调节FAO和HK-2细胞线粒体能量合成，减少细胞凋亡，抑制肾脏纤维化进程，减轻糖毒性对HK-2细胞与db/db小鼠的病理损伤。本项目对DKD的基础研究和临床防治以及基础营养学和营养治疗，具有重要的理论意义和临床应用价值。

目前国外研究发现，脂肪酸氧化（FAO）产生的能量，是高能需要肾小管上皮细胞的主要来源，FAO调节线粒体能量代谢，是上皮细胞向间质细胞转化和肾脏纤维化进程的重要因素，以FAO为靶点调控上皮细胞能量代谢，可提供治疗相关疾病基础策略。但是必需脂肪酸ALA/LA对上皮细胞向间质细胞转化的作用及其机制，国内外尚缺乏研究。本研究首先建立了糖尿病肾病的体外模型和动物模型，通过ALA/LA对糖损伤HK-2细胞的作用，发现干预剂量为50μM和100μM,混合比例为1:4的ALA/LA,可从基因和蛋白水平下调BAX，上调BCL-2，减少糖损伤HK-2细胞的凋亡。同时，50uM，1:4 ALA/LA可经TGF-β1/PPAR-α/PCG-1α通路，调节FAO和HK-2细胞线粒体能量合成，抑制肾小管细胞间质转分化。剂量浓度为500 mg/kg·d，混合比例为1：4的ALA/LA 可通过上调脂肪酸氧化关键酶CPT-1α和ACOX1mRNA和蛋白表达，提高DKD db/db小鼠肾脏的能量代谢，经由TGF-β1/PPAR-α/PCG-1α信号通路，减缓db/db小鼠肾脏纤维化的进程，减轻肾脏的病理损伤。本项目对DKD的基础研究和临床防治以及基础营养学和营养治疗，具有重要的理论意义和临床应用价值。