肝刺激因子保护SERCA活性及抑制内质网应激诱发非酒精性肝损伤的研究

Nonalcoholic steatohepatitits (NASH) is a chronic liver metabolic disorder. The prevalence of NASH is increasing extremely fast, suggesting that it is an important priority to understanding its pathogenesis. It is reported that the endoplasmic reticulum (ER) stress may contibute to the pathogenesis of NASH. While, the inactivation of sarco/endoplasmic reticulum Ca2+-dependent APTase (SERCA) is highly linked to ER stress. Recnently, we found that HSS (hepatic stimulator substane), a novel liver-specific growth factor, could alleivate ROS-induced liver damage, therefore,we attempt to investigate whether inhibition of ROS attack to SERCA by, could reduce ER stress and alleviate cell injury as well. Based upon this aim, NASH model will be reproduced in cell culture and high-fat-diet fed mice and the experimental approaches are carried out on these points. 1).Whether HSS can effectivly remove ROS and consequencely protect SERCA activity, thereby alleviating ER stress; 2). Ca2+ homostatsis, particularly in the compartment of ER-mitochondria overlap (namely MAMs), is investigated together with alternation of SERCA activity during ER stress; 3). HSS, as an antioxidant, could preserve SERCA activity at basis of recombinant HSS and HSS-transfected cells; 4). Contribution of HSS gene in prevention of NASH is going to be studied in the HSS-knockdown cells or even HSS-knockout animals. Based upon these investigations, we are hoping to illuciadate the fundemental role of HSS against ER stress and referring SERCA as a potential targeting site of NASH therapy.

非酒精性肝炎（NASH）流行日益严峻，防治刻不容缓。近报，NASH与内质网（ER）应激有关，而ER应激又与ER膜钙泵（SERCA）失活相关。我们发现，肝刺激因子（HSS）能抑制自由基致肝线粒体损伤。HSS是否可保护内质网SERCA，抑制ER应激，治疗NASH值得关注。本课题复制NASH动物-细胞模型，研究1）HSS可否减轻ER应激；减轻应激导致肝损害？2）线粒体-内质网重叠区域（称之MAMs）Ca2+失稳态乃触发ER应激之关键，故研究NASH发病时MAMs内Ca2+稳态改变，观察HSS保护SERCA活性，探讨其阻断ER应激发生的机制；3）本室已制备HSS基因敲除（KO）动物，本课题将使用HSS-KO动物验证HSS抑制ER应激效果。本计划旨在阐述SERCA失活-ER应激-NASH发病之关系，并首次利用HSS对SERCA失活这一关键靶点进行干预，为HSS作为保肝药物而应用，提供科学依据。

非酒精性脂肪性肝炎（NASH）是一种非饮酒引起的慢性脂肪性肝病，但NASH发病机制尚不明了。有报道NASH发病可能与内质网-线粒体相互作用失调有关，核心环节事件包括内质网Ca2+经线粒体相关的内质网膜（MAM）途径大量涌入线粒体，造成线粒体钙稳态，干扰脂质线粒体内的正常代谢。Ca2+是细胞内重要的信号分子，能调节包括内质网应激与细胞凋亡在内多种重要细胞功能。.在国家自然科学基金系列资助下，我们前期研究先后证明，肝再生刺激因子（HSS）能有效抑制内质网应激，减轻线粒体损伤，保护肝细胞，但其具体机制仍不十分明了。内质网（ER）作为细胞内最大的Ca2+库，通过其膜上两种蛋白分子，钙离子通道（主要为IP3R）和钙泵（主要为SERCA），负责线粒体钙释放与回收。本课题以探讨HSS维持MAM钙信号稳态为目标，探究HSS保护肝细胞的具体机制。通过MCD饮食诱导小鼠NASH模型，并且尾静脉注射腺病毒-HSS表达载体，明确NASH小鼠模型中HSS保护肝脏、减轻脂质堆积的作用。结果表明，在外源性给予HSS的NASH模型小鼠，脂质堆积显著减少，肝脏损伤明显减轻，内质网应激减轻，线粒体损伤减少。在HSS转染的细胞系中，MAM区域内的SERCA表达及活性均增加，同时IP3R表达及活性降低。实验明确了HSS通过改善MAM中SERCA的活性，抑制线粒体Ca2+超载；通过Bcl-2与IP3R相互作用抑制IP3R的活性，减少ER-Ca2+释放；通过维持细胞内钙稳态，减轻内质网应激，进而保护肝细胞，从线粒体与内质网互作的一个侧面，解释了HSS减轻脂质损伤的新机制。.鉴于HSS对线粒体的保护作用，我们探讨了HSS在脂肪肝的缺血/再灌注损伤（IRI）中保护肝脏的作用。首先制备NASH小鼠IRI损伤的动物模型，明确HSS对脂肪肝IRI损伤具有保护作用；通过细胞缺氧-复氧（H/R）模型，阐述了HSS减轻IRI氧化应激，提高抗氧化能力，减少ROS产生，进而保护线粒体功能，从而最大程度地抑制细胞凋亡，减轻NASH肝脏应对IRI的作用机制。为临床上NASH的发病机制及其治疗提供了全新的视角和治疗靶点；在脂肪肝IRI中减少肝细胞的损伤，提高肝脏的存活提供的新的思路和潜在的药物。