HIRI gradually becomes an urgent problem affecting the recovery of liver function after hepatocellular carcinoma resection. HIRI often presents severe ERS, leading to the disorders in intracellular protein folding process. The excessive accumulation of misfolded proteins in hepatocytes cannot be eliminated in time, which surely promotes the occurrence of apoptosis [19]. Studies have confirmed that IPC can protect the liver function in HIRI by inhibiting hepatocyte apoptosis, inducing autophagy or activation of related signaling pathways [20]. Autophagy can engulf harmful proteins or organelles to form vesicles, then fuse with lysosomes to degrade its contents, which is essential to maintain the metabolic balance [21]. The current study confirmed that HIRI significantly damaged hepatocytes through ERS-dependent apoptosis. However, IPC, including DIPC and RIPC, both acquired the protective effects in HIRI through activating moderate autophagy and molecular chaperones or HSPs localized in the endoplasmic reticulum. The protective effects on hepatocytes of IPC were reversed by autophagy inhibitor. Meanwhile, autophagy activation induced by IPC attenuated ERS-dependent apoptosis after HIRI both in vivo and in vitro, and the effects were mimicked by autophagy activator, suggesting that IPC could attenuate ERS-dependent apoptosis of hepatocyte through regulating autophagy in HIRI.
IPC is confirmed to be a safe and efficient method with potential protective effect on organs recently [22]. Moreover, RIPC is characterized by handling remote limbs or tissues instead of directly handling the target organs, which is easy to operate clinically. Some studies implied that three cycles of 5 minutes ischemia and 5 minutes reperfusion significantly decreased troponin I level in cardiac surgery [23, 24]. We compared the protective efficacy on liver function between RIPC and DIPC in the present study, and confirmed that they both alleviated hepatocyte injury, attenuated hepatic sinusoid congestion, vacuolization and inflammatory cell infiltration in HIRI mice. In vitro, IPC also significantly reduced the LDH level in OGD/R hepatocytes. IPC acquires two effective windows in strengthening the resistant ability of hepatocytes to HIRI. The early phase usually occurs 3–6 hours while the late phase occurs 24 hours after IPC [25]. Autophagy may play a crucial role in the early phase of IPC. As a double-edged sword in cellular activities, autophagy is essential in degrading and recycling useless proteins and organelles by engulfing substances into autolysosome, which helps to avoid harmful substances accumulation. However, excessive autophagy is associated with cell death by attacking normal proteins or organelles [26, 27].
Recent researches implied a relationship between IPC and autophagy both in myocardial cell and neurons [28, 29]. We also demonstrated that the autophagy activation mediated the protective effects of IPC on hepatocytes, at least partly. Autophagy activation was evaluated by LC3-II and p62 levels. Known as the microtubule-associated protein 1A light chain 3, LC3 consists of cytosolic type I (LC3-I) and membrane-bound type II (LC3-II). During the formation of autophagosome, LC3-I is transformed to LC3-II after splitting some amino acids at the C-terminus, so the LC3-II level usually directly reflects the number of autophagosome and autophagy activation [30]. Furthermore, p62 is also a sensitive marker to reflect autophagy flux as it is degraded by the autophagosome [31]. We found that IPC significantly up-regulated LC3-II expression and down-regulated p62 expression in HIRI mice and OGD/R hepatocytes, indicating that autophagy was activated by IPC for harmful substances cleaning. Moreover, the molecular chaperones and HSPs were also increased significantly after IPC intervention in vivo and in vitro, which is beneficial to help to eliminate harmful substances. Molecular chaperone GRP78 can regulate protein folding, translocation and secretion in endoplasmic reticulum, enhanced GRP78 activities relieved harmful protein accumulation and prevented neuronal apoptosis [32]. Also, HSPs is a crucial regulator of protein folding and translocation under stress, which could be up-regulated by unfolded proteins accumulation. HSP60 and HSP70 were well observed to acquire beneficial effect in brain ischemia and ischemic tolerance. HSP60 was observed to locate in mitochondrial matrix and could be predominantly activated by unfolded proteins accumulation in mitochondria to protect neurons from ischemic injury [33]. Studies also confirmed that HSP70 was closely associated with autophagy in eliminating unfolded proteins in neurodegenerative disorders [34].
Under HIRI, endoplasmic reticulum, mitochondria, nucleus and cytoplasm all respond differently to the unfolded proteins accumulation through specific signaling pathways. In cerebral ischemic stroke, glucose deprivation, hypoxia, reactive oxygen species or calcium overload can destroy the normal function of the endoplasmic reticulum and induce ERS [35]. Similarity to autophagy, moderate ERS is beneficial to harmful protein clearance, while excessive ERS will disrupt the balance between protein synthesis and decomposition, even promote cell apoptosis [36]. Hepatocyte firstly activates the UPR response under ERS to activate the following three endoplasmic reticulum transmembrane effector proteins PERK, IRE1 and ATF-6. PERK can phosphorylate eIF2α, resulting in the inhibition of translations of some important proteins. Continuous or excessive ERS will damage the functional metabolism or injury other organelles, even disturb gene transcription and translation in cell nucleus. These changes eventually activate an ERS-dependent apoptosis pathway, accompanied by enhanced expression of CHOP, cleaved caspase-12, and cleaved caspase-3 [37, 38]. To confirm the regulatory effect of autophagy on ERS, autophagy inhibitor 3-MA was administrated. 3-MA can inactivate class III phosphatidylinositol 3-kinases to destroy the membrane dynamics involved in autophagic vesicle trafficking, resulting in the inhibition of autophagosomes formation [39]. In the present study, we found that autophagy activation induced by IPC vitally increased HSPs and alleviated ERS-dependent apoptosis markers in vivo and in vitro, liver damage and the number of apoptotic hepatocyte were also attenuated by IPC. However, these protective effects of autophagy activation induced by IPC were reversed by 3-MA. Also, autophagy activator RAP mimicked the protective effects of autophagy on ERS-dependent apoptosis in vitro, further suggesting the regulatory effect of autophagy on ERS-dependent apoptosis. However, the precise regulatory mechanisms of autophagy on ERS-dependent apoptosis remains to be further studied.
In summary, IPC or pharmacological preconditioning can induce moderate autophagy and up-regulate some molecular chaperones to strengthen the endogenous defense mechanisms, which is beneficial to alleviate ERS-dependent apoptosis and protect hepatocytes from HIRI. These findings will provide a novel strategy for potential therapies of liver suffering from ischemia reperfusion injury.