基于Sirt1/p53/PUMA通路探讨小檗碱对脂肪肝供体肝移植后缺血再灌注损伤的保护作用

Compared with normal liver, steatotic liver is much more susceptible to ischemia/reperfusion (I/R) injury, which produces multiply risks of postoperative primary liver failure. According to the literature, PUMA is a proapoptotic p53-target gene and identified as a major mediator of I/R induced mitochondrial injury. Our previous study showed that berberine preconditioning could decrease the levels of apoptosis in steatotic grafts after liver transplantation via enhancing the activity of Sirt1 deacetylase in nuclear. But the mechanism was unclear. We hypothesize that berberine can decrease mitochondria-dependent apoptosis by activating Sirt1/p53 pathway to inhibit the transcription of PUMA, which can attenuate the I/R injury of steatotic grafts after liver transplantation. To test this hypothesis, we will build the liver transplantation using steatotic grafts model in vivo and in vitro. Then we will analyze the mechanism of berberine's protective effect by using inhibitors of Sirt1 and p53. Then we intend to investigate the regulation of Sirt1/p53 pathway on pro-apoptotic gene PUMA by site-directed mutagenesis, chromatin immunoprecipitation and luciferase report assays. The study investigates the mechanism of berberine's protective effects against I/R injury of steatotic grafts, which provides a new thought in alleviating the shortage of donor liver.

脂肪肝供体对缺血再灌注（I/R）损伤比正常肝脏更加敏感，移植术后发生原发性肝无功能的风险成倍增加。文献报道，PUMA是目前发现促凋亡作用最强的p53靶基因，参与I/R所致线粒体损伤的关键环节。课题组前期工作证实，小檗碱可提高大鼠脂肪肝供体肝移植后细胞核内去乙酰化酶Sirt1活性、减少肝细胞凋亡，但具体机制不明。我们推测：小檗碱可上调Sirt1/p53通路抑制PUMA转录，减少线粒体依赖性细胞凋亡，缓解脂肪肝供体肝移植后I/R损伤。为了验证这一假说，课题组拟建立大鼠脂肪肝供体原位肝移植模型，应用Sirt1及p53抑制剂干预，观察小檗碱对线粒体功能及细胞凋亡的影响；在大鼠肝细胞体外脂肪变性模型上，我们采用定点突变、染色质免疫沉淀、荧光素酶报告基因等技术探讨Sirt1/p53通路对PUMA转录活性的影响，进一步阐明小檗碱发挥保护效应的分子机制，为防治脂肪肝供体I/R损伤、缓解供肝短缺提供新思路。

相对于正常肝脏而言，脂肪肝供体对缺血再灌注（I/R）损伤更加敏感，术后移植物功能延迟恢复的发生率明显升高。为了提高肝移植患者术后生存率、充分利用有限的供肝资源，增强脂肪肝供体抗I/R 能力至关重要。小檗碱是一种传统中药提取物，可通过抑制氧化应激和炎症反应，减轻包括心脏、脑等脏器的缺血再灌注损伤。因此，本课题旨在探讨小檗碱对脂肪供肝缺血再灌注损伤的保护作用及具体分子机制。动物实验建立中度脂肪肝大鼠（高脂饮食12周）70%肝I/R损伤模型，I/R造模前2周给予小檗碱灌胃处理（200mg/kg/天，连续2周），体外实验建立棕榈酸诱导大鼠原代肝细胞脂肪变性联合缺氧/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、生化检测、ELISA、Western Blot等方法检测大鼠肝脏损伤程度以及线粒体损伤情况；采用MTT实验及荧光探针标记法检测缺氧/复氧对脂质化肝细胞损伤程度及活性氧的产生水平；采用Western Blot、免疫荧光等方法检测肝脏I/R和肝细胞H/R损伤过程中线粒体结构及功能损伤情况；采用Western Blot检测Sirt1/p53信号通路及细胞凋亡蛋白表达的变化。结果发现：小檗碱预处理可明显降低脂肪肝大鼠肝I/R后清转氨酶水平，肝组织损伤区域减小，活性氧产生也相对降低。进一步通过给予相应信号通路干预剂证实，小檗碱改善脂肪供肝缺血再灌注损伤的机制与促进肝细胞Sirt1表达、抑制p53及线粒体依赖性细胞凋亡有关。本课题证实，小檗碱预处理通过调控Sirt1/p53信号通路抑制肝细胞线粒体依赖性细胞凋亡，从而减轻脂肪供肝缺血再灌注损伤。