In this work, hepatic ischemia was successfully induced by 70% occlusion for 30 min followed by 2 h reperfusion. Hepatic IR resulted in hepatic injury as proved by increased level of liver enzymes. Hepatic IR causes the disruption of the membrane stability of hepatocytes due to necrosis, cellular damage, and structural changes with release of large quantities of liver enzymes [37,38,39].
Moreover, a histopathological study showed a corrupted hepatic architecture in the form of hepatocyte vacuolization and necrosis, nuclear pyknosis, lymphocytic infiltration, and congestion of blood sinusoids. These findings agree with the results of previous studies by Peralta et al. [40], Serracino-Inglatt et al. [41], and Crockett et al. [42].
The present hepatic functional and structural damage could be attributed to oxidative stress, increased ROS production, and lipid peroxidation associated with decreased antioxidant defense. This is proved by the increased plasma MDA and decreased plasma TAC. The decreased TAC could be explained by its over consumption to face the increased oxidative stress. Meanwhile, liver enzymes showed a significant positive correlation with MDA and a significant negative correlation with TAC.
This view is in line with previous reports which showed that reperfusion caused a generation of ROS that reacts with lipids in the cell membranes and initiate lipid peroxidation and is responsible for the IR injury [43, 44].
In addition, the initial stage of hepatic IR is reported to mediate the oxidative stress, the production of massive amounts of ROS, neutrophil activation, and its adherence to endothelial cells and release of proteases and finally induce death of hepatocyte [45].
In the current work, a significant positive correlation between the plasma levels of MDA and TNF-α is observed which could be explained by the ability of ROS to induce an inflammatory response via cellular signaling [46, 47]. In addition to the significant increase in the plasma level of TNF-α following hepatic IR and its significant positive correlations with liver enzymes in this study, it denotes that TNF-α could be involved as an underlying mechanism in the current hepatic dysfunction and structural damage [48].
Other studies demonstrated that IR results in increased gene expression of both nuclear factor kappa B (NF-kβ) and Toll-like receptor 4 (a protein involved in both innate and adaptive immune system response), which constantly increased TNF-α leading to liver damage [38, 49].
TNF-α has been reported to induce inflammation by several mechanisms. It interacts with other inflammatory cytokines and chemokines and activates the production of ROS [50]. In addition, TNF-α upregulates the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 on endothelial cells, activating the transcription factor; NF-kβ resulting in the production of inflammatory mediators as TNF-α, interleukins (IL); IL-1-B, IL-6, inducible nitric oxide synthase, and cyclo-oxygenase with amplification of the process of inflammation [51,52,53].
The decreased plasma level of nitrite observed in hepatic IR rats provides an additional explanation for the observed hepatic functional and structural damage. This deduced from significant increase in the serum levels of ALT and AST and their significant negative correlations with the plasma level of nitrite.
In line with this, Taha et al. [54] reported that IR injury is associated with a remarkable decrease in the bioavailability of NO, which represents an important initiating event in the pathophysiology of post-ischemic injury in a variety of different tissues, including the liver.
Diminished NO levels within liver during IR was found to be derived from both decreased production due to downregulation of endothelial nitric oxide synthase (eNOS) with hepatic IR, and increased scavenging by the elevated levels of ROS produced during reperfusion [1, 55, 56]. This scavenging causes the formation of peroxy-nitrite, the free radical, that rapidly reacts with all components such as proteins, lipids, and DNA further damaging the cell [57].
Insufficient NO production was assumed to be the main cause for vasoconstriction during reperfusion period, sinusoidal narrowing, and reduction of microcirculatory blood flow [58, 59]. Moreover, hepatic IR injury was attributed to an imbalance in the ratio of endothelin (ET) to NO, with an increase in the plasma levels of ET and a concomitant fall in the plasma levels of NO in the first few hours of reperfusion [53].
The significant negative correlation exerted between the plasma nitrite and plasma MDA in the current work suggests that the oxidative stress could be implicated in reduction of nitrite plasma level.
Pretreatment with l-Arg attenuated hepatic IR injury as evidenced by decreased liver enzymes and hepatic damage. Hepatocytes had regression of necrosis, vacuolization, and nuclear pyknosis.
In this study, the favorable effects of l-Arg on hepatic functional and structural alterations could be attributed to the increased NO level deduced from the significant increase in the plasma level of nitrite by l-Arg treatment so that it reached the control values and the associated significant negative correlations existed between plasma level of nitrite and serum levels of ALT and AST.
This is in consistence with other studies, in which pretreatment with l-Arg resulted in activation of NO synthesis, increased concentrations of NO stable metabolites nitrite, and nitrate anions in both the blood and liver tissue and suppression of increased ALT and AST activities [60]. In addition, the activation of eNOS and the production of NO by this enzyme was found to increase liver graft preservation and improve liver function after reperfusion [61, 62].
The beneficial effects of the modulation of l-Arg/NO pathway was attributed before to the increase in NO bioavailability that acts by promoting microvasculature vasodilatation, opposing vasoconstriction mediated by ET, inhibition of platelet aggregation, and adhesion, as well as the reduction of interaction between leukocytes and endothelial surface resulting in the reduction of the inflammatory activity, and inhibiting caspases to prevent apoptosis, in addition to the superoxide scavenging property and detoxification of ROS following l-Arg use [63, 64].
It seems that l-Arg mediates its protective effect in hepatic IR in the present study not only through NO, but also has direct antioxidant effect deduced from the significant decrease in the plasma level of MDA and the significant increase in the plasma level of TAC. This assumption is supported by the significant negative correlations existed between nitrite and MDA, as well as the significant positive correlation between nitrite and TAC. In line with our assumption, in a rat model of carbon tetrachloride-induced hepatotoxicity, pre- and post-treatment with l-Arg decreased the hepatic MDA content and enhanced the hepatic antioxidant enzymes; these results were attributed either to NO ability to function as scavenger or to the antioxidant effects of l-Arg itself [65].
The other protective mechanisms of l-Arg could be attributed to its anti-inflammatory mechanism. This is deduced from the suppression of TNF-α plasma level by treatment as well as the significant negative correlation between plasma levels of nitrite and TNF-α.
In agreement, NO was reported to inhibit pro-inflammatory cytokines, including TNF-α, IL-1B, IL-1α, and IL-12, which may induce inflammatory cascade during liver IR injury [8, 54].
The exercise model encountered in this study was in the form of chronic moderate swimming exercise, 2 h daily and 6 days/week for 4 weeks. This exercise training ameliorated hepatic injury and dysfunction induced by hepatic IR, as revealed by a significant decrease in the serum levels of ALT and AST and the plasma levels of MDA and TNF-α, but a significant increase in TAC and nitrite levels. Also, an improvement of hepatic morphology was demonstrated by the decreased score of necrosis, vacuolization, and congestion compared to hepatic IR group.
These findings denote that exercise training abrogated hepatic functional and structural impairment induced by hepatic IR. These results are consistent with previous studies [21, 66, 67].
Exercise training exerts more powerful anti-inflammatory and antioxidant defense effects than l-Arg with less hepatocellular injury. This is evidenced by significant decrease in plasma TNF-α and the increase in plasma TAC that reached control values and better regression of scores of vascular congestion, hepatocyte necrosis, and vacuolization. Meanwhile, the serum levels of ALT and AST and plasma levels of nitrite and MDA were insignificantly changed.
The current proposed anti-inflammatory role of exercise training is supported by other studies [66, 68,69,70]. Similarly, Dallak et al. [71] showed that swim exercise for 60 min three times per week, for 4 weeks, reduced the serum levels of inflammatory biomarker, TNF-α in a rat model of high fat diet, via reduction in visceral fat mass with a subsequent decrease in adipokine release.
The decrease in final body weight and BW % change recorded in the Ex + IR group (unpublished data) compared to the IR, L-Arg + IR, and sham groups could provide possible explanation for the reduction of plasma TNF-α. The adipose tissue was found to be able to produce inflammatory cytokines such as TNF-α and IL-6 and several potent chemo-attractant cytokines [72]. The accumulation of monocytes as macrophages in the adipose tissue is thought to be a major source of increased systemic concentrations of inflammatory cytokines [73].
Opposite to our work, the intense exercise was found to induce muscle microtraumas, increase the release of inflammatory cytokines into the bloodstream [74, 75], and lead to tissue damage with increased production of ROS and inflammatory mediators [76, 77] induce hepatic inflammation through inflammatory cell infiltration in rats [78]. This contradiction could result from the use of different intensities and durations of exercise models other than the model used in the current study.
Exercise training in the present work had protective effect not only through anti-inflammatory mechanisms but also had antioxidant effect. Other studies showed that regular exercise enhance hepatic antioxidant capacity, redox status, reduced the hepatic MDA level which reflect lipid peroxidation [66, 67, 79, 80].
On the contrary to the present results, severe exercise was found to mediate an oxidative effect and to increase the hepatic MDA levels. The authors assumed that intense exercise increases oxygen consumption and may produce an imbalance between ROS and antioxidants, inducing oxidative stress [76].
Compared to l-Arg treatment, moderate intensity exercise did not differ significantly as regards the serum levels of ALT and AST and the plasma levels of MDA and TAC but showed only a significant decrease in the plasma levels of TNF-α and nitrite which indicated that the hepatoprotective effect of moderate intensity exercise was largely dependent of its anti-inflammatory effects and only dependent partly on increasing NO.
Compared to hepatic IR, Oxy pretreatment in the Oxy + IR group resulted in improvement of hepatic dysfunction which was observed from the significant reduction in the serum levels of ALT and AST, plasma levels of MDA and TNF-α together with significant increase in TAC and nitrite and alleviated IR histopathological injury. However, such improvements did not reach the control levels. These results are in agreement of other studies [81,82,83].
The beneficial effect of Oxy against hepatic IR could be attributed to suppression of oxidative stress as confirmed by the significant decrease in plasma level MDA and the significant increase in the plasma level of TAC. In addition to its anti-inflammatory effect as seen by decrease in plasma TNF-α.
The antioxidative effect of Oxy was attributed to its ability to break lipid peroxidation chain [82, 84, 85]. While its anti-inflammatory effect was mediated by reduction of the serum TNF-α, inhibition of neutrophils migration and neutrophil-derived pro-inflammatory cytokines, parenchymal injury, and tissue inflammation [81].
Also, the increased plasma level of nitrite upon Oxy treatment in the Oxy + IR group compared to the hepatic IR group, could be a third mechanism by which Oxy can produce hepatoprotection during IR.
However, the Oxy + IR group did not differ from the l-Arg + IR group regarding the serum levels of ALT and AST and the plasma levels of MDA and TAC but only showed a significant reduction in the plasma levels of TNF-α and nitrite which indicated that hepatoprotective effect Oxy was mediated mainly through its anti-inflammatory effect of but less on its NO producing effect.
Combination of both exercise training and Oxy pretreatment in the Ex + Oxy + IR group resulted in the attenuation of the hepatic damage caused by hepatic IR evidenced by the significant decrease in serum levels of ALT and AST, and plasma levels of MDA and TNF-α together with a significant elevation in the plasma levels of TAC and nitrite and histopathological improvement.
On comparison with l-Arg, combination of both exercise and Oxy in this study displayed more pronounced hepatoprotection evidenced by the significant decrease in the serum levels of ALT and AST together with recession of histopathological injury, to a greater extent than in l-Arg. Such superiority of combined treatment over l-Arg could be attributed to their more prominent antioxidant and anti-inflammatory effects but not due to their NO-enhancing effect as the plasma levels of MDA, TNF-α, and nitrite were significantly lower than in l-Arg-treated group.
Compared to exercise only group (Ex + IR), the combination of exercise and Oxy in the Ex + Oxy + IR group exerted additive effects which offered more hepatoprotection where the levels of ALT, AST, TNF-α, and MDA were significantly lowered indicating dampening of the inflammatory response and lipid peroxidation which is independent on NO as its level did not differ significantly in both groups.