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ARTICLE
Effects of Recombinant Human Erythropoietin and 2-Mercaptoethane Sulfonate on Liver Ischemia-Reperfusion Injury in Rats

Objectives: The aim of this study was to determine the effects of recombinant human erythropoietin and 2-merkaptoethane sulfonate, administered in combination, on the biochemical and histopathological changes of ischemia-reperfusion injury.
Materials and Methods: Fifty female Wistar Albino rats were used in this study. The animals were randomly divided into 5 groups: a sham group that underwent standard laparotomy only, an ischemia-reperfusion group that was subjected to 30 minutes of hepatic ischemia and 2 hours of reperfusion, a group that intraperitoneally received 1000 IU/kg recombinant human erythropoietin 5 minutes before ischemia-reperfusion, a group that intraperitoneally received 150 mg/kg 2-merkaptoethane sulfonate 15 minutes before ischemia-reperfusion, and a combined group that received both drugs intraperitoneally before ischemia-reperfusion. After the reperfusion period, serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and malon-dialdehyde levels were measured. We also evaluated histological changes in rat liver tissues samples.
Results: Serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and malondialdehyde levels were high in the control groups, but aspartate and alanine aminotransferase levels were within normal limits, especially in rats that only received recombinant human erythropoietin. In rats that received combined treatment, parenchymal alterations in liver tissue were less severe than in the other groups and necrosis did not occur.
Conclusions: Recombinant human erythropoietin was clearly more effective than 2-merkaptoethane sulfonate for preventing oxidative injury. When the agents were combined, obvious biochemically and histologically protective effects occurred, providing significant tissue protection in ischemia-reperfusion injury.


Key words : Animal model, Liver enzymes, Liver regeneration

Introduction

Ischemia-reperfusion (I/R) injury is the damage that occurs in tissues that have remained ischemic for a specific time following reestablishment of the blood circulation. The liver is an organ that depends mainly on oxygen demand due to its high energy requirements and is therefore sensitive to anoxic or hypoxic conditions.1,2 Hepatic ischemia can be classified as cold and warm ischemia. The develop-ment of cold ischemia is linked to the preservation and storage of the organ before organ transplantation.3 On the other hand, warm ischemia is associated with shock, trauma, pancreatitis, and transplant and liver surgery, in which there may be a temporary interruption of the blood circulation.4

The hypoxia that develops in ischemia decreases the ATP production. The reduction of energy damages the active carrier pumps on the membrane. The Kupffer and endothelial cells swell as a result of intracellular osmotic pressure and electrolyte imbalance. At the same time, the increase in the amount of intracellular calcium as a result of impaired calcium hemostasis activates the phospholipases of the cellular membrane and subsequently worsens the degradation of the structure of the membrane. The activation of Kupffer cells in the early period of reperfusion leads to release of proinflammatory cytokines, such as tumor necrosis factor α and interleukin 1, and the production of reactive oxygen radicals (ROR).4 During neutrophil activation in the late period, neutrophils accumulate in the sinusoidal and post-sinusoidal venules and adhere to the endothelial cells. The ROR production and release occurring in the neutrophils cause liver damage. Liver I/R injury is associated with several processes such as activation of Kupffer cells, ROR production, neutrophil infiltration and increased levels of adhesion molecules, release of cytokines, hepatocyte damage, and separation of sinusoidal endothelial cells.5,6

Nitric oxide and endothelin 1 are also important vasoactive agents that play a role in I/R injury. Nitric oxide, which is formed from L-arginine via nitric oxide synthetase (NOS), causes vasodilation in sinusoids and in the presinusoidal region.6,7 Therefore, sinusoidal congestion is one of the histopathological changes seen in I/R injury.

2-Mercaptoethane sulfonate (MESNA) is a small synthetic molecule that has the potential to scavenge reactive oxygen species, thanks to its sulfhydryl group.8 It is primarily used to reduce hemorrhagic cystitis induced by cyclophosphamide.9 The effectiveness of MESNA as an antioxidant has been demonstrated in various in vivo and in vitro models.10 Moreover, the direct suppressive effects of thiol-containing MESNA on hydrogen peroxide production can be used as an antioxidant to limit the toxic effects of free radicals produced by any kind of oxidative injury. Recent animal studies have demonstrated its antioxidative effect against I/R injury in the kidneys,11 liver,12 and intestines.13,14 The mechanism of the antioxidative effect of MESNA is related to removal of ROR,15 enhancement of the oxidative status of tissues,16 and, more recently, inhibition of nuclear factor κB (NF-κB) activation.13 In liver I/R injury, NF-κB is activated; inhibition of this activation protects the liver.

Recombinant human erythropoietin (rhEPO) is a low molecular weight glycoprotein hormone that has been reported to exhibit beneficial effects on angiogenesis, tissue-protective effects in I/R injury, and anti-inflammatory properties. Recombinant human erythropoietin decreases leukocyte accu-mulation and the secretion of the proinflammatory cytokines tumor necrosis factor α and interleukin 6, as well as intercellular adhesion molecule 1 expression, after ischemia.17,18 The glycoprotein hormone can induce changes in NOS directly or indirectly.19 Several studies have experimentally demonstrated that rhEPO treats apoptotic liver injury after I/R injury by reducing the JNK phosphorylation and NF-κB inhibitor degradation.20

Although there are many studies in the literature that have examined the effects of various agents on I/R damage of organs (including heart, kidney, and liver), single-regimen agents are generally used to protect against I/R damage. Both rhEPO and MESNA provide effects by different mechanisms against hepatic injury. We hypothesized that the combined use of these agents, acting through different pathways, may have a more protective effect against I/R injury. Thus, in this study, we investigated the protective effects of rhEPO and MESNA against liver I/R injury.

Materials and Methods

Experimental animals
This study included 50 female Wistar Albino rats of the same age (3-4 months) and approximately equal weight (250-300 g). Animals were bred in the Uludağ University Experimental Animal Breeding and Research Center under standard conditions in rooms with the appropriate 20 °C to 22 °C temperature, humidity, and ventilation, with periods of 12 hours of light and 12 hours of dark. Animals were fed on standard rat chow with water ad libitum. The experimental study was conducted in the same center with the permission of the Uludağ University Animal Care and Use Committee Ethics Board. The study protocol was in conformity with the “Guide for the Care and Use of Laboratory Animals” (published by the National Institutes of Health 86-23, revised in 1985) and was in conformity with the ethical guidelines of the 1975 Helsinki Declaration.

Experimental protocol
Fifty rats were divided into 5 groups. In a one-way analysis of variance study, sample sizes of 8, 8, 8, 8, and 8 were obtained from the 5 groups whose means were to be compared. A total sample size of 40 achieved 84% power to detect differences among the means versus the alternative of equal means using an F test with .05 significance level. Considering the possibility of loss of rats during the surgical procedure, each group was randomly composed of 10 rats. Ten different groups were formed with 5 rats in each group. The standard diet was stopped 2 hours before the study, and only water intake was allowed. In all experimental groups, the rats were placed on the experimental table under intramuscular administration of 5 mg/kg xylazine (Romphun) and 60 mg/kg ketamine (Ketalar) anesthesia, with intermittent ketamine as needed. A standard 5-cm midline laparotomy was performed.

The hepatoduodenal ligament comprising the portal vein, hepatic artery, and the common bile duct advancing to the liver was clamped with a vascular clamp to create total hepatic ischemia for 30 minutes. The color change caused by ischemia was observed on the liver surface. After the ischemia period, the vascular clamp was opened, and the 2-hour reperfusion phase was initiated. During the reperfusion period, the skin was closed using a stapler to minimize fluid loss.

There were 2 control groups: the sham group underwent standard laparotomy only, and the I/R only group received I/R as described after a standard laparotomy. There were 3 treatment groups. (1) Group B rats received 1000 IU/kg rhEPO intraperitoneally 5 minutes before hepatic ischemia was induced and then entered into the I/R period. (2) Group C rats received 150 mg/kg MESNA intraperitoneally 15 minutes before ischemia and then entered into the I/R period. (3) Group D rats (combination group) received 1000 IU/kg rhEPO at 5 minutes before ischemia and 150 mg/kg MESNA at 15 minutes before ischemia and were then entered into the I/R period (Figure 1).

A repeat laparotomy was performed in all groups after the 2-hour reperfusion period, and the left lobes of all rat livers were placed in 10% formalin solution for pathological examination. The rats were kept under constant anesthesia during ischemia and reperfusion. The experiment was completed by cardiac puncturing, and 5 mL of blood was obtained for biochemical tests, at which time the experiment was ended. At the end of the experimental protocol, we carried out the euthanasia procedure under general anesthesia.

Biochemical analyses
The 5-mL blood samples obtained from all rats were centrifuged at 2000 revolutions/min for 10 minutes. Serum samples were then stored at -80 °C. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and gamma-glutamyltransferase (GGT) levels were measured from collected serum samples to determine liver damage. Malondialdehyde levels in the serum were evaluated as a guide for lipid peroxidation in tissues.

Histological analyses
The liver left lobe tissue samples obtained after the I/R period, which had been stored in 10% formalin, were embedded in paraffin using a tissue embedding device (Shandon Histocentre 3; Thermo Electron Corporation). Paraffin samples were cut with a sliding microtome at a thickness of 1 μm (Leica SM2000R Microtome; Leica Instruments GmbH) and stained using hematoxylin-eosin. Central vein dilatation, sinusoidal congestion, degeneration, and necrosis in the hepatocytes were examined under a light microscope. The percentiles were determined for each parenchymal change observed when grading the damage. For this reason, 25% slices were given for each histopathological change. Accordingly, cases with more than 75% observed change were regarded as severe and those under 75% were regarded as mild.

Statistical analyses
The statistical evaluation of the results was performed by the Uludağ University Biostatistics Department, for which the SPSS for Windows version 11.0 statistical package program was used. Continuous variables are presented as mean ± standard deviation or median and interquartile range (25th to 75th percentile); the categorical data are presented as frequency (number) and percentage. The Pearson chi-square test, the Fisher exact chi-square test, and the Kolmogorov-Smirnov test, Kruskal-Wallis test, and the Mann-Whitney U test were used for statistical analyses. The significance level was accepted as P < .05.

Results

Biochemical levels
In all treatment groups, the AST, ALT, LDH, GGT, and MDA values were lower than those shown in the I/R-only group. The AST and ALT values were significantly lower in the single pretreatment regimens compared with the combined regimen group, but this change was not meaningful for LDH and malondialdehyde (Figure 2). We observed that the biochemical parameter GGT was significantly lower, especially in group B rats compared with all other groups (P < .001).

Serum malondialdehyde levels
The decrease in malondialdehyde levels was significant when we compared group C versus group B (P = .035). However, there was no difference in this respect between group C and group D (P = .481).

Pathological parenchymal changes after ischemia-reperfusion injury
In our examinations of tissue changes, no sinusoidal congestion was observed in liver samples from the sham group, but sinusoidal congestion was present in almost all liver tissues of the other groups. Central vein dilatation was mostly observed in liver tissue samples from the I/R-only group. Half of the central vein changes observed in this group were severe dilatation. On the other hand, severe central vein dilatation was not found in the other groups.

No hepatocyte degeneration was observed in the sham group. Although normal hepatocyte structure was observed in only 40% of the I/R-only group, 70% of the hepatocytes in group B were not affected by I/R. On the other hand, there were no significant differences between the groups with regard to central vein dilatation, sinusoidal congestion, hepatocyte degeneration, and necrosis.

Although necrosis was not observed in any liver tissue samples from the sham group, 60% of the
I/R-only group had diffuse necrosis. In the treatment groups, only 1 tissue sample from group C had necrosis. Although necrosis was less common in group C than in the I/R-only group, this difference was not significant. Group D (combination group) had 4 liver tissue samples with necrosis (Figure 3).

Statistical significance was only present for central vein dilatation in one-to-one comparisons of the treatment groups versus the I/R-only group. Central vein dilatation was significantly less common in group D than in the I/R-only group (P = .007).

We observed that half of the tissue samples from the I/R-only group had severe parenchymal changes (>75%). Severe parenchymal damage was observed in 2 tissue samples from group B and in 3 tissue samples from group C. There was a noteworthy difference between the I/R-only group and the combined group (group D) in terms of parenchymal changes. All changes seen after I/R injury in the combined group were less than 75%. This difference in severe damage between the 2 groups was significant (P = .03).

The severely enlarged central vein seen in the I/R-only group and the widespread necrosis seen in all areas are highly pathognomonic for injury (Figure 4). However, group B and, in particular, group D (combined group) had less frequency of central vein dilatation (30% and 20%, respectively). The difference in dilatation between group B and group D was minimal.

In general, there was sinusoidal congestion in the liver tissues examined. In group B and group C, sinusoidal congestion was more common than other parenchymal changes. The hepatocyte appearance, which is the most significant share when evaluating parenchymal changes, was completely normal in 80% of the combined group (group D). Hepatocytes with significant fatty degeneration were observed in the I/R-only group and group C. Most of the liver tissues observed in group D were found to have been protected from I/R injury, except for minimal sinusoidal congestion. Necrosis was observed in some preparations, but this was very focal.

Discussion

Free oxygen radicals damage a series of biomolecules, including nucleic acids, membrane lipids, enzymes, and receptors found in tissues. At the same time, lipid peroxidation initiated by free oxygen radicals may contribute to impaired cellular function and necrosis associated with reperfusion of ischemic tissues.21 Malondialdehyde is the end product of lipid peroxidation. For this reason, it becomes meaningful to assess the glutathione-to-glutathione disulfide ratio (GSH/GSSH) and the malondialdehyde concentrations together to show oxidative stress as well as some biochemical parameters, especially in liver I/R injury.

Investigations of MESNA have suggested that MESNA inhibits the increase in malondialdehyde by clearing the reactive hydroxyl and peroxyl radicals thanks to the structure of the hydroxyl group.12 Differences in this effect on oxidative stress related to its dose and the routes of administration continue to be investigated. In a study published in 2008, MESNA was administered at different doses, both intraperitoneally and orally, during intestinal I/R. In all preischemic MESNA applications, decreased malondialdehyde concentrations and increased GSH/GSSG ratios were demonstrated, as well as a protective effect by inhibition of NF-κB activation. In the same study, intraperitoneal administration of MESNA was shown to be effective in reducing oxidative damage. Still, no differences were shown between the groups in terms of dose increase and repeated dosing. In oral MESNA administration, the relationship of this difference with dosage and time was clearly demonstrated. It has also been shown that oral administration of MESNA was more effective than intraperitoneal administration.13

Another study on the effects of MESNA on NF-κB, which plays an essential role in the early phase of liver regeneration, showed its protective effects against impaired liver regeneration in rats who had undergone hepatectomy using the Pringle maneuver. In that study, in which MESNA was delivered to rats via the intragastric oral route, the GSH/GSSH ratios and the malondialdehyde concentrations were assessed, and histopathological examination was performed. The investigators observed significantly decreased malondialdehyde concentrations and increased GSH/GSSH ratios in the group that received MESNA versus the hepatectomy, Pringle, and hepatectomy + Pringle groups. They found that there were significant decreases in AST and ALT levels in the group treated with MESNA.22

In another study showing the protective effects of MESNA against hepatic I/R injury, the effects of 150 mg/kg intraperitoneal MESNA on oxidative stress was demonstrated. In the experimental study, MESNA was delivered 15 minutes before ischemia. Although the AST and ALT levels were found to be meaningfully low in this group, the GSH level was significantly high and the malondialdehyde con-centration was meaningfully low. In the histopat-hological examination, hepatocyte and central vein appearances were observed to be normal in most areas in the group treated with MESNA. In the MESNA treatment group, the significant increase of malondialdehyde, which is the end product of lipid peroxidation, was attributed to the ability of MESNA to scavenge free oxygen radicals.12

The effectiveness of rhEPO in preventing apoptosis has been demonstrated in studies of myocardial I/R injury. In a study in rats that investigated rhEPO at different doses, rhEPO was effective in liver I/R damage by reducing the disruption of NF-κB inhibition and JNK phosphorylation. In particular, groups that received intraperitoneal rhEPO at 3 different times and different doses were compared, with examination of apoptotic cells, intrahepatic caspase 3 levels, and biochemical parameters (AST, ALT, and LDH levels). Although there was a significant decrease in these parameters in all rhEPO groups compared with the I/R group, there were no differences among the different rhEPO treatment groups. Therefore, the investigators concluded that the rhEPO-mediated protective effect was dose independent.20

In our study, we observed that malondialdehyde, the end product of lipid peroxidation, showed a more significant decrease compared with the other groups with the use of MESNA. It has been suggested that the protective role of MESNA in I/R injury is through prevention of lipid peroxidation by scavenging of free oxygen radicals. The reason for this effect having been to a lower extent in the combined group compared with the MESNA group can be explained by the fact that the I/R injury in group B was through a different pathway.

In our study, although AST and ALT levels in the treatment groups indicated a significantly lower I/R damage versus that shown in the I/R-only group, this decrease was more noticeable in group B (which received only rhEPO treatment before I/R). In group B, the more significant decrease in AST and ALT values, which are biochemical markers of liver hepatocyte cell damage, may appear as an answer for the role of rhEPO in the prevention of apoptosis.

When the pathological parenchymal changes were examined, it was once again observed that hepatocyte degeneration was least common in group B and group D, which had both received rhEPO, whereas necrosis was least common in the group C, which did not receive rhEPO. Oxygen radical-initiated lipid peroxidation may contribute to necrosis associated with reperfusion of ischemic tissues. The group that only received MESNA treatment, which probably works by scavenging the ROR, appears to be the group with the least necrosis compared with the other treatment groups. The main effect of MESNA pretreatment is on necrosis, not on sinusoidal occlusion and hepatocyte degeneration. On the other hand, we suggest that MESNA prevented the deterioration in cellular functional and necrosis caused by oxygen radical-initiated lipid peroxidation; therefore, group C had a lower extent of these presentations compared with the I/R-only group, although the difference only trended toward significance. In another study, similar histopathological results were found with regard to the effects of MESNA on liver I/R injury in rats. In this study, the MESNA-treated I/R group showed well-preserved liver parenchyma, with mild sinusoidal dilatation, and the central vein and hepatocytes appeared normal in most areas.12

Because rhEPO has positive effects on angiogenesis and causes changes in NOS directly or indirectly, it has more positive effects, especially with regard to hepatocyte degeneration and central vein dilatation. When MESNA, which did not have a positive effect on these pathological changes, was administered together with rhEPO, a significant improvement in pathological changes was observed, which was not seen when MESNA was administered alone.

In the assessment of biochemical parameters in our study, we observed that the single treatment regimens provided a statistically significant decrease in some parameters compared with the rat group that received both rhEPO and MESNA (group D). However, considering the pathological changes, group D had significantly more preserved liver parenchyma than the I/R-only group, especially in terms of severe pathological changes. Again, central vein dilatation was observed at a statistically lower extent in group D, which had received combined therapy, compared with the I/R-only group.

Although rhEPO and MESNA use different pathways, significant pathological and biochemical results were obtained in group D compared with the I/R-only group, since both agents affect the NF-κB pathway.


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DOI : 10.6002/ect.2021.0425


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From the Department of General Surgery, School of Medicine, Uludağ University, Bursa, Turkey
Acknowledgements: The authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no declarations of potential conflicts of interest.
Corresponding author: Pinar Tasar, Bursa Uludağ University, School of Medicine, Department of General Surgery, Gorukle, Turkey
Phone: +90 5324814960
E-mail: pinartasar@gmail.com