Sirt6通过RACK1调控IRE1α改善胆汁淤积性肝损伤的机制研究

Biliary atresia (BA) is a common cause of cholestasis, and the mechanism by which induced liver injury has not yet been elucidated. IRE1α is important in the process of reducing liver injury in the endoplasmic reticulum (ER) stress pathway, and its activity is regulated by RACK1. Our studies found that the expression level of Sirt6 was up-regulated in the liver tissues of BA patients and bile duct ligation mice, and the ER stress pathway was activated. In vitro and in vivo studies confirmed that knockdown of Sirt6 can aggravate liver injury caused by bile acid, the phosphorylation level of IRE1α was up-regulated, RACK1 directly interacts with IRE1α, and RACK1 expression is regulated by Sirt6. These results suggest that the Sirt6/RACK1/IRE1α pathway plays an important role in the regulation of cholestatic liver injury. This study will use adenovirus infection, RNA-seq, co-immunoprecipitation, mass spectrometry and other techniques to focus on the role of Sirt6 in improving cholestatic liver injury, and elucidate its role and molecular mechanism in regulating RACK1 activity or expression and affecting endoplasmic reticulum stress, in order to provide a new target for the treatment of cholestatic liver injury.

胆道闭锁是胆汁淤积的常见原因，其引起肝损伤的机制尚未阐明。内质网应激通路中IRE1α在减轻肝损伤过程中十分重要，其活性受RACK1调节。本项目前期研究发现，在胆道闭锁患者和胆管结扎小鼠肝组织中Sirt6的表达水平适应性上调，且内质网应激通路被激活。体内外实验证实，敲降Sirt6可加剧胆汁酸引起的肝损伤，上调内质网应激通路中IRE1α的磷酸化水平，RACK1与IRE1α直接互作，且RACK1表达受Sirt6调控。这些结果提示Sirt6/RACK1/IRE1α途径在调节胆汁淤积性肝损伤中发挥着重要作用。本课题将利用腺病毒感染、RNA-seq、免疫共沉淀和质谱分析等技术，重点明确Sirt6在改善胆汁淤积性肝损伤中的作用，阐明其调控RACK1活性或表达而影响内质网应激的作用和分子机制，以期为胆汁淤积性肝损伤的治疗提供新靶点。

胆道闭锁是胆汁淤积的常见原因，其引起肝损伤的机制尚未阐明。内质网应激通路中IRE1α在减轻肝损伤过程中十分重要。氨酰tRNA合成酶（aaRSs）是蛋白质生物合成的关键酶，而特定aaRSs的紊乱与特定疾病的病因有关。甘氨酰tRNA合成酶（GARS）的显性突变与各种神经退行性疾病相关，但其在肝病中的作用尚未研究。本项目前期研究发现，在胆道闭锁患者和胆管结扎小鼠肝组织中Sirt6的表达水平下调，且内质网应激通路被激活。体内外实验证实，敲降Sirt6可加剧胆汁酸引起的肝损伤，上调内质网应激通路中IRE1α的磷酸化水平，共免疫沉淀分析发现，胆汁酸处理增加了IRE1α的乙酰化，该乙酰化被Sirt6的过度表达所消除，并被Sirt6抑制剂OSS_128167所增强。此外，胆汁淤积的肝脏中GARS水平显著升高。肝脏特异性GARS敲除小鼠的肝脏损伤明显减少。GARS缺陷通过影响肌醇需要酶1α（IRE1α）和C/EBP同源蛋白（CHOP）和拼接X盒结合蛋白1（XBP1s）的磷酸化，减轻了胆管结扎（BDL）诱导的内质网（ER）应激。此外，GARS通过上调转染因子E2F1来降低Sirt6的水平。我们的研究揭示了GARS通过上调E2F1抑制Sirt6表达而激活ER应激促进导胆汁淤积性肝损伤中的关键作用。