基于AMPK/Sirt1通路介导的自噬探讨小檗碱对移植肾缺血再灌注损伤的保护作用及机制

Ischemia reperfusion (I/R) injury can lead to graft dysfunction or nonfunction after kidney transplantation. According to the literature, Sirt1 induced autophagy has protective effects against renal I/R. Our previous studies have found that allograft kidney transplantation of rats would lead to the up-regulation of oxidative stress and apoptosis. Autophagy and AMPK activity would be inhibited. Berberine could attenuate the oxidative stress level by promoting autophagy. But the related mechanism remains unclear. We hypothesize that berberine can improve the deacetylation of autophagy-related factor via activating AMPK/Sirt1 pathway, promote autophagy and inhibit mitochondria-dependent apoptosis after kidney transplantation; To test this hypothesis, we will build the allogeneic kidney transplantation model of rats and analyze the mechanism of berberine's renoprotective effect by using the inhibitors of AMPK and Sirt1. Then we intend to investigate the regulation of AMPK/Sirt1 pathway on autophagy related gene by site-directed mutagenesis, immunoprecipitation and luciferase report assays. Our study investigates berberine's protective mechanism via regulating AMPK/Sirt1 axis induced autophagy, which provides a new thought in preventing I/R injury after kidney transplantation.

缺血再灌注(I/R)损伤可导致肾移植术后移植物功能延迟恢复甚至丧失。文献报道，Sirt1介导的自噬对肾脏I/R损伤具有保护作用。课题组前期研究发现，异体肾移植大鼠肾脏氧化应激及细胞凋亡率升高，细胞自噬及AMPK活性均降低；小檗碱预处理可上调细胞自噬、减少肾脏氧化应激，但具体机制不明。我们推测：小檗碱可通过上调AMPK/Sirt1通路促进自噬相关转录因子去乙酰化，增强细胞自噬，抑制线粒体依赖性细胞凋亡，减轻移植肾I/R损伤。为此，课题组拟建立大鼠异体肾移植模型，采用AMPK及Sirt1抑制剂干预，观察小檗碱预处理对肾脏细胞自噬及线粒体功能的影响；在缺氧/复氧诱导原代大鼠肾小管上皮细胞损伤模型上，我们采用基因定点突变、免疫沉淀、荧光素酶报告基因检测等方法探讨AMPK/Sirt1通路对自噬相关转录因子去乙酰化的影响，进一步阐明小檗碱发挥肾脏保护效应的分子机制，为肾移植I/R损伤的防治提供新思路。

肾脏缺血再灌注 (I/R) 可造成肾移植患者术后早期移植物功能延期恢复甚至无功能，增加早期死亡的风险。小檗碱（berberine，BBR）是一种异喹啉生物碱，具有抗炎、降血脂、治疗糖尿病等药理作用，最新研究发现，BBR可减轻肾I/R损伤，但是具体机制尚未完全阐明。 动物实验建立大鼠异体肾移植模型，I/R造模前1周给予小檗碱灌胃处理（100mg/kg/天，连续1周），体外实验建立大鼠肾小管上皮细胞缺氧/复氧（H/R）损伤模型，于H/R造模前给予小檗碱预处理6h。分别采用HE染色、Masson染色、生化检测、Western Blot等方法检测大鼠肾脏损伤程度；采用MTT实验及ELISA法检测缺氧/复氧对肾小管上皮细胞损伤程度及氧化应激水平；采用Western Blot、流式细胞术等方法检测移植肾I/R和肾小管上皮细胞H/R损伤过程中细胞凋亡情况；采用Western Blot及免疫共沉淀法检测Sirt1/FoxO3α信号通路蛋白在胞浆及细胞核的分布以及FoxO3α乙酰化的改变。结果发现，小檗碱预处理可明显降低大鼠异体肾移植后血清肌酐及尿素氮水平，肾脏组织病理学损伤减轻，氧化应激水平也相对降低。进一步通过给予相应信号通路干预剂证实，小檗碱改善移植肾缺血再灌注损伤的机制与促进肾小管上皮细胞Sirt1表达、促进FoxO3α入核后去乙酰化有关。本课题证实，小檗碱预处理通过调控Sirt1/FoxO3α信号通路抑制肾小管上皮细胞凋亡，从而减轻移植肾缺血再灌注损伤。