PPAR-γ激活对肾缺血再灌注损伤后肾小管上皮细胞自噬的调控研究

Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is a type of ligand activated nuclear transcription factors and involved in a variety of pathological processes. Recent studies showed that the activation of PPAR-γ regulates autophagy of tumor cells and neurons after cerebral ischemia reperfusion injury. However, the regulation of autophagy in renal ischemia-reperfusion injury (IRI) is not yet clear. Our previous studies have shown that the activation of PPAR-γ by pioglitazone hydrochloride could protect from renal IRI, and a large number of studies confirmed regulation of autophagy of renal tubular epithelial cells after renal IRI played a protective role, therefore, we hypothesized that PPAR-γ activation could regulate autophagy of renal tubular epithelial cells after renal IRI. This project is proposed to establish renal IRI in rats and hypoxia/reoxygenation model of renal tubular epithelial cell, to study the changes of autophagy-lysosome by transmission electron microscopy, PCR and western blot methods, to clarify the regulation function of autophagy after PPAR-γ activation on renal tubular epithelial cells after kidney IRI; Using cell model in vitro and animal model in vivo, to study protein expression of autophagy signal pathway by western blot method, and further to clarify molecular mechanism of autophagy by PPAR-γ activation regulating renal tubular epithelial cells after kidney IRI, in order to search the new target on prevention and treatment of renal IRI.

过氧化物酶体增殖物激活受体-γ(PPAR-γ)是一类配体激活型核转录因子，参与了体内多种病理生理过程。新近发现，PPAR-γ激活调控肿瘤细胞和脑缺血再灌注损伤后神经元自噬。然而，其对肾缺血再灌注损伤(IRI)自噬的调控尚未清楚。我们的前期研究表明，PPAR-γ激活剂盐酸吡格列酮对肾IRI起保护作用，而大量研究证实肾IRI后调控自噬对肾小管上皮细胞具有保护作用，因此，我们推测PPAR-γ激活可能调控肾IRI后肾小管上皮细胞的自噬。本课题拟建立大鼠肾IRI和肾小管上皮细胞缺氧/复氧模型，通过透射电镜、PCR、免疫印迹等方法研究自噬溶酶体活性的变化，阐明PPAR-γ激活对肾IRI后肾小管上皮细胞自噬的调控作用；利用体外细胞模型和体内动物模型，通过免疫印迹法研究自噬信号通路蛋白的表达变化，进一步阐明PPAR-γ激活调控肾IRI后肾小管上皮细胞自噬的分子机制，以期探寻防治肾IRI的新靶点。

本研究通过建立大鼠肾缺血再灌注损伤模型，应用PCR、免疫印迹等方法检测了盐酸吡格列酮预处理大鼠肾IRI后24h肾组织中自噬相关蛋白LC3、Beclin-1、P62、AMPK等的表达以及凋亡蛋白caspase-3、caspase-8的表达；同时通过体外肾小管上皮细胞NRK-52E缺氧4h/复氧12h模型，同样检测了细胞上述自噬相关蛋白和凋亡蛋白的表达。主要研究结果：1.盐酸吡格列酮可以降低肾IRI后血清肌酐和尿素氮水平；减少肾组织中细胞的坏死与凋亡；2.盐酸吡格列酮升高肾组织中PPAR-γ表达；3. 盐酸吡格列酮升高肾组织中自噬相关蛋白LC3‖、Beclin-1表达；4. 盐酸吡格列酮升高肾组织中AMPK表达;5. 经盐酸吡格列酮预处理的肾小管上皮细胞NRK-52E缺氧4h/复氧12h，可以升高自噬相关蛋白LC3‖、Beclin-1表达，升高AMPK表达，降低mTOR表达。上述结果表明，盐酸吡格列酮激活PPAR-γ对肾缺血再灌注损伤后肾小管上皮细胞自噬可以起到调控作用。本研究进一步从细胞自噬角度阐明PPAR-γ激活保护肾缺血再灌注损伤的分子机制，初步探寻了防治肾缺血再灌注损伤的新靶点。