Hepatic I/R can contribute to severe liver damage and is a major clinical problem during liver surgical procedures. Studies of the pathophysiology and underlying mechanisms of liver I/R injury have yielded a number of potential therapeutic alternatives (Gracia-Sancho et al. 2015). However, effective therapeutics in rodent models are limited, and no pharmaceutical therapies specifically target I/R-induced liver injury have been approved for human use. Thus, preventing and attenuating hepatic I/R injury is an unmet clinical need. In this study, we confirm that MaR1 protects against deterioration of liver function in a mouse liver I/R model. The mechanism of hepatoprotection of MaR1 involved anti-inflammatory, anti-apoptosis and anti-oxidative effects during hepatic I/R injury. We show here that MaR1 acts via a known signaling partner, ALXR, to enhance activation of Akt with downstream effects on inflammatory responses and apoptosis resulting in alleviation of I/R-mediated liver damage (Fig. 7).
Previous studies investigating in vivo effects of MaR1 in rats used doses of 4 ng/g body weight given intraperitoneally 1 h prior to liver I/R (Soto et al. 2020). However, we investigated the effects of different doses of MaR1 given at the beginning of reperfusion via the tail vein. We found that a dose of MaR1 of 20 ng/mouse, effective to suppress I/R injury in mice. Thus, the use of MaR1 at the time of I/R could be therapeutically useful.
Numerous studies have provided strong evidence that inflammation-driven by reperfusion-mediated responses is the major contributor to liver damage in I/R injury (Jiménez-Castro et al. 2019), (van Golen et al. 2012). Increased levels of circulating inflammatory cytokines are associated with greater liver I/R injury (Liu et al. 2019). Therefore, recent efforts for therapeutic strategies have focused on the direct suppression of inflammation at the reperfusion stage (Datta et al. 2013, (Selzner et al. 2003). MaR1 has a series of pro-resolving actions including improved macrophage phagocytosis, diminished neutrophil infiltration and reduced pro-inflammatory cytokine release (Serhan et al. 2009, (Buckley et al. 2014). Consistent with these previous investigations, we found that treatment with MaR1 significantly decreased the serum IL-1β and IL-6 levels after hepatic I/R injury. Moreover, there were reduced Ly6G-positive inflammatory cells in MaR1-treated mouse livers. Similarly, we demonstrated that MaR1 negatively regulates ERK signaling, a pivotal component regulating inflammation and cell death, both in vivo and in vitro following liver I/R injury. Taken together, these results demonstrate that MaR1 effectively modulates inflammatory responses after liver I/R. We speculate that this may occur, in part, through the suppression of cellular death.
In addition to inflammation, recent evidence indicated that apoptosis is another essential contributor to hepatic I/R injury (Guo et al. 2020). Apoptosis can also be enhanced by reperfusion-induced inflammatory responses (Jassem et al. 2019). We confirmed that MaR1-treated mice exhibited decreased hepatocyte apoptosis compared with controls after liver I/R. However, this is contradictory to previous findings in a rat liver I/R model, where protein expression of cleaved caspase-3 was dramatically increased with MaR1 treatment (Soto et al. 2020). The reason for the contradictory results is unclear and may be related to species differences, time of treatment with MaR1, or potential adverse/toxic effects of MaR1 in the previously-published study.
In this study, we are the first to demonstrate that the protective role of MaR1 on hepatic I/R injury is independent of NPCs, which suggests that MaR1 has a direct effect on HCs. Hepatic I/R injury is a complicated pathological state, in which oxidative stress also exerts a critical role (Yi et al. 2020). During the reperfusion period, HCs, neutrophils and macrophages can produce ROS, which can trigger the activation of the inflammatory immune responses (Jaeschke 2011). Furthermore, the excess generation of ROS, leading to protein and DNA damage through lipid peroxidation, is regarded as a major cause of oxidative damage to cellular membranes during I/R injury (Rani et al. 2016). In our study, the level of tissue MDA, a main product of lipid peroxidation, was markedly decreased in the MaR1-treated mouse livers following hepatic I/R insult. The eradication of reactive free radicals is dependent on many different antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT), which keep the balance between antioxidative effects and oxidative stress responses (Sun et al. 2017). Loss of antioxidant enzymes causes accumulation of free radicals, which further exacerbates I/R-induced injury (Han et al. 2016). Our study reveals a novel effect of MaR1 on the formation of MDA potentially via increased antioxidative enzyme (e.g. GPX) activity during hepatic I/R injury. Thus, it is reasonable to assume that the beneficial effects of MaR1 on liver I/R injury are, at least, partly due to maintenance of the balance between antioxidative and oxidative stresses.
It has been widely accepted that the SPMs, such as MaR1, exert anti-inflammatory actions through direct binding to their corresponding G protein-coupled receptors (GPCRs) (Serhan 2010). However, due to the complicated cellular context, each receptor is capable of interacting with more than one SPM. Previous studies suggested that MaR1 could interact with ALXR (lipoxin A4 receptor) in CLP-induced sepsis in mice to induce protection (Gu et al. 2018). Other studies revealed that resolving E1, another SPM, could induce the generation of endogenous lipoxin A4 in the lung, which was similarly protective (Haworth et al. 2008). In the present study, we confirmed a role for ALXR in mediating the protective effects of MaR1 in liver I/R, although this could be via acting directly at ALXR, or induction of lipoxin A4 production via another MaR1-induced signaling pathway. Further study will be needed to determine which pathway dominates.
Akt is a downstream effector of PI3K signaling shown to modulate multiple cellular effects (Martin et al. 2005). Akt signaling is a known regulator of liver I/R injury (Li et al. 2019), and there is increasing evidence that Akt signaling initiates cell survival through inhibition of apoptosis and improving cell viability (Tsuruta et al. 2002). Our studies showed that MaR1 increases Akt activation/phosphorylation after I/R injury and this was dependent on ALXR signaling. More importantly, Akt upregulation and activation were critical for MaR1-mediated hepatoprotection in liver I/R injury, which is also in line with published studies (Sun et al. 2015, (Izuishi et al. 2006).
In conclusion, our present study provides evidence that MaR1 exerts a protective role in I/R-induced injury by reducing the inflammatory response and alleviating hepatocyte apoptosis via ALXR/Akt signaling. These observations broaden our deeper understanding of the direct regulatory role of MaR1 on hepatic I/R insult. Significantly, MaR1 is an endogenous chemical mediator with few identified side effects, suggesting that MaR1 has the potential to be used therapeutically in a wide range of human diseases induced by I/R injury.