Brg1正调控STAT1/PUMA通路介导的细胞凋亡在肝缺血再灌注损伤中的作用研究

Hepatic Ischemia-reperfusion Injury (HIRI) is one of the main contributors to poor prognosis of patients who underwent hepatic surgery. The mechanisms of HIRI remains to be elucidated and effective therapeutic intervention are still being called for. Apoptosis plays an important role in HIRI. PUMA is a pro-apoptotic factor. STAT1 is a transcription factor which act as a pro-apoptotic signal. Brg1 associated chromatin remodeling complex can regulate the transcription and expression of multiple genes, and participate in many pathophysiological process. Our previous research has shown in the liver tissue of rats underwent HIRI, Brg1, STAT1 and PUMA were upregulated, along with elevated mRNA levels, poor liver function and pathological changes. In Brg1 KO mice, however, the liver function and pathological changes were significantly restored, and the protein expression and mRNA levels of Brg1, STAT1 and PUMA were all lowered. Therefore, we hypothesize that the regulation of STAT1/PUMA pathway-induced apoptosis by Brg1 is the key to HIRI. Our current research intend to apply certain genetic modification techniques such as adeno-virus transfection, Chip analysis and dual luciferase reporter assay, to establish in vitro and in vivo ischemia-reperfusion/hypoxia-redox injury model, and elucidate the mechanisms of Brg1 in regulating STAT1/PUMA induced apoptosis and the impact of this pathway on HIRI to provide new therapeutic target to HIRI.

肝缺血再灌注损伤（HIRI）是肝脏缺血手术病人不良预后的主要原因，细胞凋亡在HIRI发展中扮演重要角色，机制复杂仍未完全阐明。PUMA是促凋亡关键因子，STAT1是新近发现与凋亡有关核转录因子，但STAT1与PUMA关系仍不明确；Brg1是染色质重塑复合物重要亚基，因增强转录效能。我们前期研究证明小鼠HIR引起明显细胞凋亡；预实验发现HIR后肝Brg1、STAT1和PUMA表达上调而P53变化不显著，在Brg1基因敲除小鼠HIRI模型中这些现象明显改善；据此，我们设想Brg1正调控STAT1/PUMA介导的细胞凋亡是肝缺血再灌注损伤的重要机制。本项目拟用Brg1基因敲除鼠HIRI和肝细胞缺氧复氧模型，结合质粒转染过表达、基因沉默和Chip等技术，通过在体实验和细胞学研究论证三者的相互关系及对细胞凋亡的影响，以验证这一假说，阐明HIRI的机制，为防治HIRI提供新的干预靶点及理论依据。

肝缺血再灌注损伤（HIRI）是肝脏缺血手术病人不良预后的主要原因，活性氧在HIRI发展中扮演重要角色，机制复杂仍未完全阐明。近年发现核因子E2相关因子2（Nrf2）是一种重要的转录因子及细胞防御反应调节器，HO-1和NQO1是常见的Nrf2靶基因；Brg1是染色质重塑复合物重要亚基，能增强转录效能。Brg1是否参与了Nrf2介导的抗氧化酶诱导性表达并在HIRI的发生发展中发挥关键作用，目前仍不清楚。丙泊酚是临床常用的静脉麻醉药，在HIRI即刻使用丙泊酚进行后处理对HIRI是否具有保护作用，以及其相关的可能机制是否与Brg1、Nrf2及其靶基因的改变有关，目前仍未见报道。.因此，本研究在明确HIR后肝脏损伤以及Brg1、Nrf2和其靶基因表达动态变化的基础上，建立肝细胞缺氧复氧模型研究Brg1是否依赖Nrf2对肝细胞Nrf2靶基因HO-1和NQO1转录调控的作用和机制，进而构建Brg1过表达的转基因小鼠，在小鼠HIR模型中进一步探讨Brg1在HIRI中的作用和机制；在上述基础上，通过在体、体外和临床研究探讨丙泊酚后处理能否通过激活Brg1/Nrf2通路促进Nrf2靶基因表达从而对HIRI起保护作用。.研究发现：.1. HIR对导致肝脏病理和氧化损伤，同时Brg1和Nrf2及其介导的抗氧化酶下降，而恢复Brg1表达可以增强Nrf2介导的HO-1表达以有效地增强抗氧化能力对抗肝细胞损伤。.2. 给予丙泊酚后处理可以明显改善HIRI导致的肝脏病理、功能和氧化损伤，明显增强肝组织Brg1、Nrf2 、HO-1和NQO1蛋白表达，高剂量组效果更佳。在7.5～60μM浓度范围内，丙泊酚后处理可以明显增强H12R4导致的L02细胞存活率和抑制LDH释放。采用siRNA干扰技术沉默Brg1和Nrf2会逆转丙泊酚后处理减轻肝细胞损伤及减少ROS生成的作用；敲除Brg1后会显著降低Brg1、Nrf2和HO-1的蛋白表达，对NQO1的表达无明显作用；而沉默Nrf2会显著降低Brg1、Nrf2、HO-1和NQO1的蛋白表达。对肝移植患者给予丙泊酚后处理可以明显改善HIRI导致的肝脏病理和功能损伤，明显增强肝组织Brg1、Nrf2 、HO-1和NQO1蛋白表达。.3. 过表达Brg1可以改善HIR诱导的肺氧化损伤，而该保护作用可能通过Nrf2通路起作用。