缺血预处理诱导自噬参与保护大鼠脂肪肝热缺血再灌注损伤作用的机制研究

Steatotic livers are more susceptible to ischemia/reperfusion (I/R) injury during liver surgery. Ischemic preconditioning (IPC) has been shown to attenuate I/R injury in steatotic livers in recent years. However, the potential mechanisms involved the protective effects of IPC are not fully understood. In our preliminary experiments, we demonstrated that: (1) IPC attenuated hepatic warm I/R injury via induction of autophagy; and (2) autophagy level was reduced in steatotic livers. Prompted by these results, we hypothesized that decreased autophagy could contribute to the increased susceptibility of steatotic livers to warm I/R injury, and autophagy induced by IPC could protect liver against I/R injury through clearance of damaged mitochondria (mitophagy). In the present study, we will use an in-vitro model of I/R in primary cultured steatotic hepatocytes and an in-vivo model of liver warm I/R to demonstrate that: (1) the correlation of autophagy level and increased susceptibility of steatotic livers to warm I/R injury, (2) the effect of autophagy induced by IPC on mitochondrial damage and hepatocellular injury during I/R; and (3) the effect of autophagy on hepatocellular injury during I/R. Finally, we will explore the protective mechanism of IPC in hepatocellular damage in experimental I/R injury in-vitro in isolated steatotic hepatocytes as well as in-vivo. Modulation of autophagy signaling may be an important novel therapeutic strategy in the setting of I/R.

脂肪肝对热缺血再灌注(I/R)损伤比正常肝脏更为敏感，缺血预处理（IPC）可以减轻脂肪肝I/R损伤，但具体的作用机制尚不明确。我们前期研究表明：（1)IPC通过诱导自噬减轻正常肝脏热I/R损伤；（2）自噬水平在脂肪肝中显著降低。因此，我们推测：（1）脂肪肝对热I/R损伤的高敏感性可能与自噬水平低下有关；（2）IPC通过诱导自噬，清除受损的线粒体（线粒体自噬），从而减轻脂肪肝I/R损伤。本课题拟应用体外培养脂肪肝细胞I/R损伤细胞模型，和MCD+HF饲料诱导的大鼠脂肪肝热I/R损伤动物模型，进一步探索：（1）自噬与脂肪肝I/R损伤的相关性；(2)抑制IPC诱导的自噬，研究IPC对脂肪肝大鼠肝脏I/R损伤的影响；（3）给予调控自噬干预，研究自噬对脂肪肝大鼠肝脏I/R损伤的影响。通过上述研究我们将阐明IPC诱导自噬在脂肪肝大鼠肝脏I/R损伤中的保护效益，为脂肪肝I/R损伤防治提供新的思路。

脂肪肝对热缺血再灌注(I/R)损伤比正常肝脏更为敏感，缺血预处理（IPC）可以减轻脂肪肝I/R损伤，但具体的作用机制尚不明确。我们前期研究表明: (1) IPC通过HO-1上调自噬减轻正常肝脏热I/R损伤；(2)自噬水平在脂肪肝中显著降低。因此，我们推测：(1) 脂肪肝对热I/R损伤的高敏感性可能与自噬水平低下有关；(2)IPC通过HO-1上调自噬，清除受损的线粒体（线粒体自噬），从而减轻脂肪肝I/R损伤。在我们的研究中，我们发现：(1) 肝细胞脂肪变可显著下调细胞自噬活性， 可能机制为：肝细胞脂肪变性导致HO-1表达减低，下调其对calpain2活性的抑制作用，从而上调calpain2对自噬蛋白的降解作用，最终导致细胞自噬活性减低；(2) 脂肪肝对热I/R损伤的高敏感性与自噬水平低下有关; (3) IPC显著减轻线粒体损伤及脂肪肝脏I/R损伤，并且可以上调细胞自噬活性；(4) 通过自噬抑制剂3-MA抑制IPC诱导的细胞自噬，可以显著减弱IPC对线粒体损伤及脂肪肝脏I/R损伤的保护作用， 通过自噬诱导剂rapamycin诱导细胞自噬，可以显著减轻线粒体损伤及脂肪肝脏I/R损伤。这些证据提示IPC可以通过诱导细胞自噬从而保护脂肪肝脏I/R损伤。进一步机制探讨发现：(1) IPC可以上调HO-1的表达及下调calpain2 的活性；(2)抑制IPC诱导的HO-1可以显著抑制calpain2 活性，抑制IPC诱导的细胞自噬，并且可以显著减轻IPC对线粒体损伤及脂肪肝脏I/R损伤的保护作用；(3)抑制Calpain2 的活性可显著诱导细胞自噬，减轻线粒体损伤及脂肪肝脏I/R损伤。这些证据提示，IPC可以通过上调HO-1，抑制Calpain2 对自噬蛋白的降解作用，上调细胞自噬活性，减低线粒体损伤，从而保护脂肪肝脏I/R损伤。通过上述研究我们阐明了IPC诱导自噬在脂肪肝大鼠肝脏I/R损伤中的保护效益，为脂肪肝I/R损伤防治提供新的思路。