Sepsis is a systemic inflammatory response to infection and generates harmful consequences, such as multiple organ damage and death. Despite significant improvements in its treatment, sepsis still accounts for 26.7% and 41.9% of all deaths in hospitals and intensive care units, respectively [1]. Primary sepsis treatment involves source control with antibiotics; however, in recent years, modulating host inflammatory responses (i.e., using corticosteroids, prostaglandins, and leukotriene inhibitors) has garnered considerable research interest. Although several novel treatment modalities have been introduced as prospective sepsis treatment options, none are definitive and thus further research is required [2].

The pathophysiology of sepsis-induced multi-organ damage is extremely complex. Uncontrolled systemic inflammatory responses and ischemia/reperfusion (I/R) processes related to low cardiac output, arterial hypotension, microcirculation failure, and mitochondrial dysfunction are believed to be contributing factors [3–5]. Specifically, the liver contributes to inflammatory responses by generating proinflammatory cytokines and acute phase proteins [6,7]. Therapeutic interventions reducing sepsis-induced inflammatory cytokines and hepatic dysfunction caused by cytokines may reduce multiple organ damage and mortality risks in sepsis [8].

Remote ischemic conditioning (RIC) is manifested by several cycles of brief, reversible, and non-lethal mechanical blood flow occlusion of distal tissue or organs, with reperfusion exerting protective effects on distant target tissue or organs. RIC can be performed either before ischemia (remote ischemic preconditioning [RIPC]) or after ischemia (remote ischemic postconditioning [RIPoC]) [9]. Several clinical trials have suggested that RIC is a promising approach that protects multiple organs (e.g., brain, heart, and kidneys) against the deleterious effects of I/R, even though inconsistencies exist in the literature [10–12]. Although the precise mechanisms underpinning these protective effects remain unclear, the effects are associated with downregulated systemic inflammatory and oxidative stress responses. Although few studies have investigated the effects of RIPC or RIPoC in sepsis, the application and therapeutic efficacy of RIPoC toward hepatic dysfunction in sepsis remain unknown.

Therefore, to address this, we examined if RIPoC ameliorated liver injury via anti-inflammatory and antioxidant marker levels in a lipopolysaccharide (LPS)-induced sepsis rat model. 

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In protocol 1 analyses, serum ALT and AST levels were higher in the L+2H, L+6H, L+12H, and L+18H groups when compared with the control group (P < 0.001), but no significant differences were identified between the L+12H and L+18H groups (Fig. 2A). For MDA and SOD measurements that reflected cellular lipid peroxidation caused by oxidative stress no significant changes in the L+2H group were observed when compared with the control group, whereas in the L+6H, L+12H, and L+18H groups, MDA was significantly higher and SOD lower when compared with the control group (P < 0.05). When compared with the L+12H group, similar results were observed in the L+18H group (Figs. 2B and C). TNF-α and NF-κB levels were higher in the L+2H, L+6H, L+12H, and L+18H groups when compared with the control group (P < 0.05) but they plateaued after 6 h (Figs. 2D and E).

In protocol 2 analyses, serum ALT and AST and hepatic tissue MDA levels were lower, whereas SOD activity was higher in the L+6R+18H and L+12R+18H groups when compared with the L+2R+18H group (P < 0.001). ALT and AST levels were significantly different between the L+6R+18H and L+12R+18H groups (P < 0.001) (Fig. 3A); however, no significant differences were recorded for MDA levels and SOD activity (Figs. 3B and C).

In protocol 3 analyses, serum ALT and AST levels in the L+2R+6H group were significantly lower when compared with the L+2R+12H and L+2R+18H groups (P < 0.001); however, no differences were observed between the L+2R+12H and L+2R+18H groups. In the L+6R+12H group, liver enzyme levels were lower when compared with the L+2R+12H group (P < 0.001) that were sampled at the same time but postconditioned at different times (Fig. 4A). MDA levels were lower and SOD activity was higher in the L+2R+6H group when compared with the L+2R+12H and L+2R+18H groups (P < 0.001). When compared with the L+2R+12H group, MDA levels were low and SOD activity was high in the L+6R+12H group (P < 0.001) (Figs. 4B and C).

In protocol 4 analyses, serum ALT, AST, and MDA levels were lower while SOD activity was higher in the RIPoC group (subjected to RIPoC four times at regular intervals) when compared with the control group (P < 0.05) (Figs. 5A-C). Furthermore, TNF-α and NF-κB levels in the RIPoC group (western blotting) were lower when compared with the control group (P < 0.05) (Figs. 5D and E). In the HE-analyzed liver tissue, the degree of PMN infiltration in the RIPoC group was lower when compared with the control group (Fig. 6A). Intrahepatic sinusoidal neutrophil numbers were significantly decreased in the RIPoC group when compared with the control group numbers (Fig. 6B).

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We showed that RIPoC stimulus via transient hind limb ischemia significantly alleviated LPS-induced liver injury in a septic rat model. In all the RIPoC treatment groups, serum ALT and AST levels were reduced, indicating that RIPoC protected cells against liver injury. RIPoC appeared to function as an immunological and antioxidant modulator in sepsis based on significant reductions in inflammatory and oxidative stress markers after its application [18].

RIC is an interesting and clinically significant tissue conditioning modality. The techniques is minimally invasive and may be applied to transplantation, vascular surgery, and cardiac surgery settings [19]. Brief I/R cycles at distant tissue may protect organs when subjected to potentially lethal ischemia [20,21]. Short IR cycles in distant tissue release humoral factors such as interleukins (IL), adenosine, opioids, bradykinin, and nitric oxide (NO) [22]. Under ischemia, these humoral factors and direct stimuli are detected by remote organ innervation [23]. These protective signals may activate G-protein-coupled receptors or receptor tyrosine kinases that induce the reperfusion injury salvage kinase (RISK) pathway, or inflammatory cytokines via glycoprotein 130 or the TNF receptor to activate the survivor activating factor enhancement (SAFE) pathway [24]. Although the exact molecular mechanisms underpinning these protective actions remains unclear, accumulating evidence now suggests that they suppress inflammatory TNF-α and NF-κB gene expression [25], decrease reactive oxygen species (ROS) production, and suppress human neutrophil adhesion and phagocytosis. Such altered immune profile have facilitated their investigation and application in sepsis. Honda et al. [8] reported that RIC improved cardiac output and survival in a LPS-induced sepsis model. Kim et al. [26] also observed that RIPoC alleviated inflammatory responses and increased survival rates by modifying NF-κB-mediated cytokine expression in a LPS-induced sepsis model. Previous studies also reported that using RIPC or RIPoC strategies in a sepsis setting reduced inflammatory response while increasing survival; however, it is unclear if RIPC or RIPoC increased hepatic cell anti-oxidative defenses. Our data suggested that RIPoC improved hepatic function by increasing not only anti-inflammatory outputs in hepatic cells but also antioxidant defenses.

The liver is the primary site for bacterial clearance; it induces inflammatory responses to sepsis by producing acute phase proteins and proinflammatory cytokines [6,7]. NF-κB is a key transcription factor that regulates proinflammatory cytokine genes (e.g., TNF-α, IL-1β, and IL-6), adhesion molecules, chemokines, and monocyte chemotactic protein-1 [27]. Therefore, NF-κB is activated early in response to sepsis in injured organs [26]. The NF-κB pathway blockade remediates the pathological abnormalities induced by sepsis in animal models while inhibited NF-κB activation reduces multiple inflammatory gene expression, preventing multiple organ damage and increasing survival rates after sepsis. Therefore, effective NF-κB inhibition or reduced TNF-α levels are significant therapeutic objectives in sepsis treatment [28]. In the sepsis models, higher NF-κB and TNF-α levels are associated with higher mortality rates and poor clinical outcomes. Our data indicate a marked elevation in the NF-κB and TNF-α levels after LPS intraperitoneal injection, while a marked reduction in TNF-α levels was observed after RIPoC.

In sepsis, bacteria and endotoxins directly act on phagocytes that cause lipid peroxidation in cellular membranes and severe damage to cellular structure and function [29]. Free radical production and oxidative stress in ischemia cause tissue damage that is accompanied by activated inflammatory responses. Previous research reported that oxidative stress is one of the key mechanisms implicated in hepatic ischemia damage processes [30]. MDA and SOD are two major pathophysiological markers used to assess free radical metabolism [31]. MDA is an oxidative stress biomarker induced by lipid peroxidation and is the end-product of polyunsaturated fatty acid [32]. MDA levels also reflect the degree of cell injury from the reactive oxygen metabolites [33]. SOD is a key endogenous antioxidant enzyme that scavenges ROS and helps maintain mitochondrial functional integrity [17]. In our study, SOD activity in hepatic tissue was significantly decreased whereas MDA levels were significantly increased in LPS-induced sepsis rats when compared with controls. Thus, RIPoC satisfactorily reduced oxidative stress. Furthermore, the anti-inflammatory and anti-oxidative stress effects of RIPoC were putatively correlated with liver protection. The simultaneous reduction in NF-κB and TNF-α levels, and also improved oxidative stress profiles after RIPoC administration, suggested complex pathway interplay leading to liver protection. Further molecular and genetic level studies are required to fully understand this mechanistic complexity.

RIC, with different insult timing, has been used in different clinical settings. Some studies have used preconditioning that induces ischemic preconditioning prior to the ischemic insult. In our study, RIC was performed after sepsis, the RIPoC approach. In clinical settings, preconditioning before septic insult is often unfeasible, although both types of ischemic conditioning may be beneficial. To establish optimal RIPoC timing, RIPoC was performed at three different time points (2 h, 6 h, and 12 h after insult), and inflammatory and oxidative stress indicators assessed at 18 h post insult. The 12 h time point was associated with greater liver protection when compared with the 6 h time point. Also, to verify if improved anti-inflammatory and antioxidant defenses sustained responses, anti-inflammatory and antioxidant parameters were measured at 6 h, 12 h, and 18 h after the insult after performing RIPoC at 2 h after insult. When RIPoC was conducted at 2 h after insult, no improvements were observed at 12 and 18 h, indicating that RIPoC only temporarily improved liver anti-inflammatory and antioxidant parameters. This finding was consistent with Costa et al. [22] who demonstrated that RIC functioned via the temporary and short-term enhancement of liver and kidney cell antioxidant defenses to avoid the deleterious consequences of future IR injury. These observations are important for the clinical translation of RIPoC because RIPoC-induced protection may be transitory and limited-duration events. Therefore, the effects of repeated RIPoC episodes on molecular outcomes were also examined. Reports on the effects of repeated RIPoC have increased in recent years. Wei et al. [34] were the first to report that performing RIC daily for a month after myocardial infarction decreased peri-infarct inflammatory responses and enhanced cardiac functional recovery and survival. Honda et al. [8] reported that repeated RIC exerted additional benefits with respect to mortality during sepsis. Consistent with previous studies, the rats in our study subjected to repeated RIPoC demonstrated improved hepatic function when compared with control rats. Furthermore, repeated RIPoC improved anti-inflammatory and antioxidant profiles. Histological liver section examinations from LPS-induced sepsis rats revealed infiltrated neutrophils, vacuolization, and necrotic hepatocytes, while these histological changes were attenuated by repeated RIPoC. However, the mechanisms underpinning repeated RIPoC applications have not been fully elucidated in previous reports and in our study; therefore, future studies are required to determine the precise mechanisms.

Our study had some limitations. For example, increasing anti-inflammatory and antioxidant capacity levels are not the only effects expected upon RIC. RISK and SAFE pathway activation, NO synthase activity, and NO release are variables that change with RIC, but they were not analyzed in our study. Moreover, survival rates were also not assessed in this study. Nevertheless, we provided a scientific foundation and a viable functional model for RIPoC-induced hepatic protection during sepsis, implying that RIPoC could be a potential therapeutic approach for sepsis treatment.

In conclusion, we comprehensively demonstrated that RIPoC attenuated liver injury in an LPS-induced sepsis model by modifying inflammatory responses and oxidative stress response for a limited period. Although additional repeated RIPoC improved hepatic injury during sepsis, further studies are required to fully understand the additional benefits and specific molecular mechanisms underpinning repeated RIPoC.

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