SIRT6-NRF2信号轴调控APAP所致的肝细胞氧化应激损伤及修复的作用与机制研究

Hepatocyte death and subsequent liver regeneration determine the prognosis of acetaminophen (APAP)-induced liver injury, making it possible for treatment to regulate mitochondrial oxidative stress and liver regeneration. Therefore, it is important to identify suitable therapeutic targets with dual functions. Deacetylase SIRT6 was found to have a potential to regulate DNA repair, cell proliferation and oxidative stress in recent years. Our previous studies revealed that SIRT6 protein expression was significantly up-regulated at the stage of liver injury regression following by down-regulation at an early stage after APAP administration in mice. However, the detailed regulation and involved mechanism of SIRT6 is not yet clear. We also found that the expression and nuclear accumulation of NRF2 was significantly increased following APAP treatment, together with up-regulation of the expression levels of NRF2 target genes at the later stage. Therefore, we hypothesizes that SIRT6 may inhibit hepatic oxidative stress induced by APAP and promote liver repair through regulating NRF2. SIRT6 over-expressed hepatocytes and Sirt6 deficient hepatocytes, together with Sirt6-/- mice, were used to verify the role of SIRT6 on APAP hepatotoxicity. Further, whether SIRT6 regulates NRF2 and the involved mechanisms will be investigated from three aspects of mRNA, protein and transcriptional activity. This study may provide data support for SIRT6 served as a new therapeutic target of APAP induced liver injury and provide a new strategy for treating this disease.

扑热息痛(APAP)所致肝损伤的预后取决于肝细胞死亡和肝再生之间的平衡，其治疗可通过干预线粒体氧化应激和肝再生实现，故寻找具有双重调控作用的治疗靶标意义重大。近年来，去乙酰化酶SIRT6被报道能调控DNA损伤修复、细胞增殖和氧化应激。我们前期研究发现，在APAP肝毒性启动和发展阶段，小鼠肝脏SIRT6表达下调，而在损伤修复期表达显著增加，具体的调控作用和机制尚不明确。此外，APAP处理后NRF2表达和入核量增加，其下游基因的表达在损伤后期明显上调。结合文献报道，我们推测SIRT6可能通过调控NRF2抑制APAP所致肝细胞氧化应激损伤并促进组织修复。本项目拟构建SIRT6高表达和低表达肝细胞，并引入基因敲除小鼠评价SIRT6在APAP肝毒性中的作用；再从基因、蛋白和转录活性方面探讨SIRT6调控NRF2的机制，为SIRT6成为APAP所致肝损伤的新靶点提供理论依据，为该疾病的治疗提供新思路。

扑热息痛(APAP)的过量服用是导致药源性肝损伤的首要原因。氧化应激和代偿性肝细胞增殖是APAP肝毒性的两个重要环节，两者间平衡决定了APAP所致肝损伤的预后。最近研究发现，SIRT6能抵抗氧化应激相关DNA损伤，但SIRT6是否调控APAP肝毒性尚不清楚。.本项目通过转染Sirt6 siRNA干扰序列，构建沉默Sirt6的AML12小鼠肝细胞，考察沉默Sirt6的AML12细胞经APAP处理后ROS、LDH、GSH和SOD水平；同时考察AML12细胞沉默Sirt6并经APAP处理后细胞周期、细胞集落形成、细胞增殖相关蛋白表达的情况。检测沉默Sirt6的AML12细胞经Dox和APAP处理后细胞LDH水平。用qPCR、双荧光素酶报告基因和免疫共沉淀技术验证p53、NRF2与SIRT6及其下游靶基因的调控关系。.我们研究发现，沉默Sirt6的AML12细胞经APAP处理后细胞ROS生成和LDH含量显著增加、GSH和SOD含量显著降低，G2/M阻滞、集落形成能力减弱和细胞增殖相关蛋白下调。此外，沉默Sirt6后Nrf2及其下游基因Gstα/µ、Ho-1的mRNA表达下调，显著降低NRF2下游基因GSTμ和NQO1的转录激活；CoIP结果进一步显示SIRT6与NRF2存在蛋白-蛋白相互作用。.激动p53后ROS生成减少，增加NRF2核转位，使Nrf2及其下游基因Mrp2/3/4、Gstα/µ、Nqo1的表达上调，但p53无法直接激活NRF2下游基因GSTμ和NQO1的转录活性。沉默Sirt6经Dox和APAP处理后LDH升高，Dox对APAP所致肝细胞损伤的保护作用减弱。敲除p53后Sirt6 mRNA表达下调。报告基因实验结果显示，p53在转录水平调控SIRT6的表达，CoIP结果进一步表明p53与SIRT6存在蛋白-蛋白相互作用。.综上所述，SIRT6通过与NRF2转录共激活调控下游基因转录和表达，减轻APAP所致的氧化应激损伤并促进肝细胞增殖；p53调控NRF2和SIRT6的表达，并促进NRF2的核转位和上调其下游相关基因的表达。p53抵抗APAP所致肝细胞损伤的作用依赖于SIRT6。本研究揭示SIRT6调控APAP所致肝细胞损伤的作用和新机制，并阐明p53与SIRT6-NRF2信号轴的关系，为APAP所致肝损伤的临床治疗提供新策略和新思路。