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Effect of Obestatin on Hepatic Injury Induced by Renal Ischemia/Reperfusion in Rat
* Author for correspondence
Abstract
Background and Aims:
Oxygen free radicals and cytokines are considered to be important components involved in the pathophysiological tissue alterations observed during Ischemia/Reperfusion (I/R). Based on the anti-oxidant and anti-inflammatory effects of Obestatin (OB), we investigated the putative protective role of OB against I/R-induced oxidative remote organ injury.
Materials and Methods:
Male albino rats were subjected to either sham operation or bilateral renal artery clamping for 45 min and reperfusion for 24 h to induce I/R damage. Obestatin was administered intraperitoneally twice a day at a dose of 8 nmol/kg. At the end of the experimental procedure, the rats were decapitated and hepatic tissue were removed for biochemical analyses of: Malondialdehyde (MDA), an end product of lipid peroxidation; the activity of antioxidant enzymes Superoxide Dismutase (SOD) and Catalase (CAT); the Myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration and the level of pro-inflammatory cytokines (TNF-α and IL-1β). The serum Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT) and Lactate Dehydrogenase (LDH) levels were measured to assess liver function and tissue damage, respectively. Pathological histology was also performed.
Results:
The results revealed the occurrence of I/R- induced oxidative organ damage, as confirmed histologically and evidenced by an increase in the MDA level and MPO activity, and a decrease in activity of SOD and CAT. Furthermore, serum AST, ALT, LDH levels, and tissue cytokines were elevated in the renal I/R group as compared to the sham operated control group. On the other hand, obestatin treatment succeeded to modulate these observed abnormalities resulting from I/R as indicated by the reduction of MAD and the pronounced improvement of the investigated biochemical and antioxidant parameters.
Conclusion:
Since obestatin administration reversed these oxidant responses, it seems likely that obestatin has a protective effect against oxidative organ damage induced by I/R.
Keywords
Obestatin (OB)
Ischemia/Reperfusion (I/R)
Oxidative Remote Organ Injury
Introduction
Acute Kidney Injury (AKI) is a common complication during major surgeries and is associated with increased risk of chronic kidney disease and mortality1. However, the morbidity and mortality from AKI is very high and remains virtually unchanged for the past 50 years in part due to a high incidence of extra-renal complications1,2. In particular, hepatic dysfunction is very frequent in patients suffering from AKI. Furthermore, development of liver injury in patients with AKI frequently leads to other extra-renal complications including intestinal barrier disruption, respiratory failure and the systemic inflammatory response syndrome with the eventual development of sepsis and a multi-organ failure3–5. These extra-renal systemic complications secondary to AKI are the leading causes of mortality in the intensive care unit6. Indeed, clinical studies show that patients with isolated AKI have significantly better prognosis than patients with AKI plus extra-renal organ dysfunction7.
Ischemia reperfusion is a frequently encountered phenomenon in organisms. Prolonged ischemia followed then by reperfusion results in severe oxidative injury in tissues and organs8. Renal Ischemia Reperfusion (RIR) is a common cause of AKI8. RIR injury occurs in many clinical situations, such as transplantation, partial nephrectomy, sepsis, hydronephrosis, or elective urological operations10. Mortality during AKI is largely due to extra renal manifestations11,12.
Liver injury is one of the distant organ damage induced by RIR. Acute renal failure associated with liver disease is a commonly encountered clinical problem of varied etiology13. It is believed that IR injury induces inflammatory response, that elicits tissue damage in a number of organs in which reactive oxygen and nitrogen species play a key role in the pathophysiology of RIR injury13,14. It demonstrated that RIR injury may cause liver oxidative stress and increase lipid peroxidation in liver tissue15. The liver tissue of rat decreases antioxidant enzyme activities after R IR is well reported16.
Obestatin is an interesting but controversial gut hormone17. Obestatin is a circulating 23-amino-acid peptide, encoded by the same gene as ghrelin18. It is predominantly produced in the stomach, and exhibits a wide range of peripheral effects including inhibition of food intake, body weight gain, gastric emptying and regulation of jejunal motility18,19. Interestingly, several previous studies have demonstrated the protective and regenerative effects of the preproghrelin gene-derived peptides, including obestatin and ghrelin in the gastrointestinal tract, kidney and heart20–22. Likewise, a positive effect of obestatin has also been observed in the pancreas, where the peptide exhibited protective action in cerulein-induced pancreatitis23.
The anti-oxidant and anti-inflammatory effects of obestatin were previously demonstrated24–26. As the main mechanism of RIR injury is via inflammation and oxidative stress, this study was designed to evaluate the possible protective effect of obestatin on oxidative liver injury (distant organ) induced by RIR in rats.
Materials and Methods
Experimental Animals
All animal procedures were approved by the ethical committee of Faculty of Medicine, Tanta University. Thirty male Albino rats in the range of 230–280 g body weight were used in this study. All subjects were kept in an animal room of Physiology Department of Faculty of Medicine, Tanta University, in a controlled temperature and 12:12 h light/dark cycle with free access to food and water.
The animals were divided into 3 groups of 10 animals each.
Group-1: the sham group (sham operated, no I/R).
Group-2: the vehicle group (renal I/R rats treated with normal saline).
Group-3: the ghrelin group (renal I/R rats treated with obestatin).
Surgical Procedure
Rats were placed on a warming pad and anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneally). Body temperature was maintained at 37±1 ℃. A tracheotomy was performed to facilitate free breathing. The tail vein was cannulated for infusion of 0.9% NaCl solution. A midline laparatomy was performed and the renal arteries were carefully separated from around the tissues.
In the I/R groups, renal arteries were occluded by a non-traumatic micro-vascular clips for 45 min, followed by 24 h reperfusion. Occlusion was approved visually by color change of the kidney to a paler shade and reperfusion by blushing. Sham operated animals underwent identical surgical treatment, including isolation of both renal arteries. However, artery occlusion was not performed. At the end of the experimental procedure, the animals were decapitated and trunk blood samples were collected to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels as indicators of liver function and damage, respectively. Liver samples were taken and stored at −70°C for the measurement of Pro-inflammatory cytokines (TNF-α and IL-1β) and malondialdehyde (MDA) levels, superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) activities were measured. Additional liver samples were fixed with 10% formaldehyde for histopathological evaluation.
Administration of Obestatin
Rat obestatin was obtained from Yanaihara Institute (Shizuoka, Japan). Treatment with obestatin was started 24 hours after the beginning of renal reperfusion. Obestatin was administered intraperitoneally twice a day at a dose of 8nmol/kg. This dose of obestatin was determined from a previous model of injury27. An equal volume of the saline was injected into the vehicle rats. The sham group of animals only underwent laparotomy.
Estimation of Liver Function and Damage
Serum AST, ALT and LDH activities were determined to assess the liver damage by using AST, ALT and LDH (Roche Diagnostic, Mannheim, Germany) commercial kits in a Roche- Hitachi Modular Auto analyzer (Roche Diagnostic).
Estimation of Cytokine Concentrations
TNF-α and IL-1β levels were determined by using a commercially available rat ELISA kit (MedSystems Diagnostics GmbH, Vienna, Austria) and the results were expressed as pictograms per milligram protein.
Estimation of Lipid Peroxidation and Antioxidant Enzymes
Malondialdehyde (MDA) was determined in liver homogenates using commercial kits (LPO-585) from Bioxytec (Bioxytec, Portland, USA). Superoxide dismutase (SOD) and catalase (CAT) activities were measured according to Sun et al.28 and Luck29 respectively.
Determination of Myeloperoxidase Activity
Tissue samples (0.2–0.3 g) were homogenized in 10 vol of ice-cold potassium phosphate buffer (50 mmol/L K2HPO4, pH 6.0) containing hexadecyltrimethylammonium bromide (HETAB; 0.5%, w/v). The homogenate was centrifuged at 30,000 g for 10 min at 4 °C, and the supernatant was discarded. The pellet was then rehomogenized with an equivalent volume of 50 mmol/L K2HPO4 containing 0.5% (w/v) HETAB and 10 mmol/L ethylenediaminetetraacetic acid (EDTA, Sigma). MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine.2HCl. One unit of enzyme activity was defined as the amount of the MPO present per gram of tissue weight that caused a change in absorbance of 1.0/min at 460 nm and 37 °C30.
Histopathological Evaluation
For light microscopic examinations, liver samples were fixed in 10% neutral buffered formalin solution. The tissues were embedded in paraffin. The paraffin blocks were cut in 5 μm thick. The sections were stained with Hematoxylin-Eosin (H&E). All tissue sections were examined microscopically for the characterization of histopathological changes by an experienced histologist in blind fashion.
Statistical Analysis
All the values are expressed as mean ± SEM. Statistical significance between more than two groups were tested using one-way ANOVA followed by the Bonferroni multiple comparisons test using computer based fitting program (Prism, Graphpad 5). Differences were considered to be statistically significant when P < 0.05.
Results
Effect of Obestatin on Liver Function and Damage
Renal IR-induced liver injury caused significant increases in the ALT and AST levels indicating impairment of liver functions. Similarly, LDH activity, as an index of generalized tissue damage was also found to be increased compared with the control. When obestatin was administered 24 hours after the beginning of renal reperfusion, these elevations were significantly depressed (P < 0.05) (Table 1).
| Groups | Measured parameters | ||
|---|---|---|---|
| ALT (U/L) | ALT (U/L) | LDH (U/L) | |
| Sham group | 47 ± 4.7 | 82 ± 6.2 | 811 ± 67+ |
| Vehicle group (RIR +saline) | 255 ± 9.7* | 218 ± 14.5* | 1332 ± 139* |
| Obestatin group (RIR+OB) | 121 ± 5.3+ | 130 ± 6.5+ | 533 ± 92+ |
Data are expressed as mean ± S.E.M.
↰+P < 0.05 compared with vehicle group.
Effect of Obestatin on Lipid Peroxidation and Antioxidant Enzymes
The liver tissue MDA content in the sham operated control group was elevated by renal I/R injury (P < 0.05); however, obestatin treatment significantly decreased the renal I/R-induced elevation in liver MDA level (P < 0.05; Table 2). In accordance with this, renal I/R caused a significant decrease in the activities of hepatic anti-oxidant enzymes (SOD) and (CAT) (P < 0.05) compared with sham operated control group, while in the obestatin-treated renal I/R group, activities of hepatic anti-oxidant enzymes (SOD) and (CAT) was found to be preserved significantly (P< 0.05), not significantly different from that of the control group (Table 2).
| Groups | Measured parameters | ||
|---|---|---|---|
| MDA (nmol/ mg-protein) |
SOD (U/ mg-protein) |
CAT (U/ mg-protein) |
|
| Sham group | 6.15 ± 1.14* | 82.6 ±7. 8 | 17.93 ± 2.08 |
| Vehicle group (RIR +saline) | 6.15 ± 1.14* | 49.7 ± 16.8* | 12.34 ± 1.43* |
| Obestatin group (RIR+OB) | 3.77 ± 1.12+ | 73.8 ± 12.2+ | 16.39 ± 1.25+ |
Data are expressed as mean ± S.E.M.
+P↰< 0.05 compared with the vehicle group.
Effect of Obestatin on Cytokine Concentrations
Pro-inflammatory cytokines (TNF-α and IL-1β) levels were significantly higher in the liver tissue of the vehicle saline-treated I/R group (P < 0.05) than that of the sham- operated control group (Table 3). On the other hand, obestatin treatment in the I/R group decreased the elevations in levels of pro-inflammatory cytokines (TNF-α and IL-1β) significantly (P < 0.05).
| Groups | Measured parameters | |
|---|---|---|
| TNF-α (pg/ mg) | IL-1β (pg/ mg) | |
| Sham group | 1.2 ± 0.3 | 11.3 ± 1.3 |
| Vehicle group (RIR +saline) | 7.4 ± 0.8* | 18.3 ± 1.2* |
| Obestatin group (RIR+OB) | 4.2 ± 0.6↰+ | 9.4 ± 0.6+↰ |
Data are expressed as mean ± S.E.M.
+P ↰< 0.05 compared with the Vehicle group.
Effect of Obestatin on Myeloperoxidase Activity
Myeloperoxidase activity, which is accepted as an indicator of neutrophil infiltration, was significantly higher in the liver tissue of the vehicle saline-treated I/R group (P < 0.05) than that of the sham - operated control group (Table 4). On the other hand, obestatin treatment in I/R group significantly decreased liver tissue MPO level (P < 0.05).
| Groups | Measured parameters |
|---|---|
| Myeloperoxidase (U/g) | |
| Sham group | 10.8 ± 0.5 |
| Vehicle group (RIR +saline) | 19.1 ±1.2* |
| Obestatin group (RIR+OB) | 14.3 ± 1.1+ |
Data are expressed as mean ± S.E.M.
+p < 0.05 compared with the Vehicle group.
Histopathological Study
In the vehicle group (RIR + saline), areas of hepatocyte necrosis in the liver parenchyma, lymphocytic infiltration, expansion of sinusoids and scattered congestion were detected. Hepatocyte damage was not observed in the liver parenchyma of the obestatin group (RIR + OB) except for scattered necrotic hepatocytes. Expansion of blood sinusoids was less compared to the vehicle group (RIR + saline). These observations are illustrated in (Figure 1).
![The effect of obestatin treatment in the liver of rats (Hematoxylin and Eosin stain). (A) Sham group, (B) Vehicle group (RIR + saline), (C) Obestatin group (RIR + OB) [H&E X100].](/content/154/2020/5/1/img/JHSR-5-007-g001.png)
- The effect of obestatin treatment in the liver of rats (Hematoxylin and Eosin stain). (A) Sham group, (B) Vehicle group (RIR + saline), (C) Obestatin group (RIR + OB) [H&E X100].
Discussion
The present study demonstrated that Obestatin (OB) administration in RIR-induced hepatic injury improved the liver functions and decreased the elevations in serum LDH activity and pro-inflammatory cytokine levels. Furthermore, increased myeloperoxidase activity and hepatic lipid peroxidation level and decreased activity of antioxidant enzymes that were observed as the consequences of oxidative injury were also reversed by obestatin treatment. Histopathologic findings also supported the anti-inflammatory effects of obestatin in RIR-induced hepatic damage.
Ischemia/reperfusion injury is a complex process involving numerous intracellular signaling pathways, mediators, cells, and pathophysiological disturbances; its prevention during surgery is of utmost importance31. It has been suggested that I/R triggers a series of reactions mainly in the organ which is clamped and reperfused, and these reactions elicit a systemic inflammatory response by the release of cytokines and inflammatory mediators (tumor necrosis factor-α, interleukin-6, nitric oxide, etc.) which cause the generation of free radicals32,33. Inflammatory mediators released as a consequence of reperfusion activate endothelial cells and circulating neutrophils in remote organs that are not exposed to the initial ischemic insult. This distant response to I/R can result in leukocyte-dependent microvascular injury that is characteristic of multiple organ dysfunction syndrome34,35. Park et al demonstrated that acute kidney ischemia causes small intestinal generation of IL-17A and subsequent intestinal injury (villous endothelial apoptosis, epithelial necrosis, increased pro-inflammatory cell translocation and cytokine flux to the liver). These events cause hepatic injury (inflammation, apoptosis and necrosis) with increased generation and release of TNF-α and IL-6 systemically causing further multi-organ injury and systemic inflammation36. As a result, in this study, although the liver tissue specimen was not exposed to ischemia directly, we aimed to investigate whether I/R caused remote organ injury as a result of a systemic effect.
Several studies have demonstrated that ischemia and reperfusion of the tissue to be associated with lipid peroxidation, which is an autocatalytic mechanism leading to oxidative destruction of the cellular membranes, and their destruction can lead to the production of toxic, reactive metabolites and cell death37. Lipid peroxidation, as a free radical-generating system, has been suggested to be closely related to I/R-induced tissue damage, and MDA is a good indicator of the rate of lipid peroxidation. In the present study, the levels of MDA significantly increased due to I/R. This observation correlates with previous studies, in which the elevated levels of lipid peroxidation products increased from 40% to 100% above the basal values13,38.
Our results show that obestatin caused a significant inhibition in MDA production, thus indicating a reduction in lipid peroxidation and cellular injury. This result agrees with previous findings showing that obestatin induced reduction of the increased level of MDA in n a rat model of colitis24,39. Similarly, Koyuncuoğlu et al.39 demonstrated that obestatin reduced seizure-induced lipid peroxidation and oxygen-derived radicals in brain. Likewise, Ersahin et al.40 showed that obestatin administration significantly decreased MDA level in the subarachnoid haemorrhage-induced oxidative injury in rats.
Reactive Oxygen Species (ROS), such as superoxide anion, hydroxyl radical, and hydrogen peroxide, have a causal relationship with oxidative stress. Antioxidant enzymes such as SOD and CAT represent protective response against oxidative tissue-damage. SOD converted superoxide anion into H2O2. Catalase metabolize H2O2 to water. Maintaining the balance between ROS and antioxidant enzymes is therefore crucial, and could serve as a major mechanism in preventing damage by oxidative stress . In the present study, we observed a decrease in the activities of SOD and CAT in liver induced by RIR. This decrease may be attributed to oxidative inactivation of enzyme protein by lipid peroxyl radicals and excess ROS generation. Treatment with obestatin showed an improved effect on the activities of antioxidant enzymes examined as compared to vehicle group. Our results confirm previous data concerning the antioxidative properties of obestatin in other tissues24,40.
The pro-inflammatory cytokines, TNF-α and IL-1β, are known to play pivotal roles in the pathogenetic mechanisms of remote organ injury42. Following experimental AKI, hepatic TNF-α, IL-6 and IL-17A are increased36,43. Knockout mice for TNF-α, IL-17A, and IL-6 displayed reduced hepatic injury after renal ischemia, as did wild-type mice treated with antibodies to the cytokines36. Ceranowicz et al.23 reported that administration of obestatin reduces the severity of cerulein-induced acute pancreatitis, by significantly raising the pancreatic blood flow and reducing the proinflammatory IL-1β release. Similarly, El-malkey and Abdel moety revealed that administration of obestatin maintains a balance in oxidant-antioxidant status through the increase in endogenous antioxidants and the decrease in pro-inflammatory mediators in rat model of Chron’s disease24. In accordance with these observations, the findings of the current study show that obestatin treatment ameliorated RIR-induced oxidative hepatic injury by a mechanism that involves an inhibitory effect on the release of inflammatory cytokines.
Liver dysfunction and cell injury induced by neutrophils have been demonstrated in a number of experimental models, including ischemic reperfusion injury44. Several methods have been used to define the role of neutrophils in the tissue injury. One of them is to measure the activity of neutrophil-specific enzyme, MPO, which is released as a response to various stimulatory substances45. In the present study, increased hepatic MPO activity due to RIR indicates that liver injury involves the contribution of neutrophil infiltration. Since obestatin depressed the tissue MPO activity in concomitant with its anti-inflammatory effect, it appears that the mode of action of obestatin treatment in hepatic injury involves its inhibitory effect on tissue neutrophil infiltration that limits neutrophil-derived oxidative tissue damage.
In the present study following I/R injury, plasma ALT, AST, and LDH levels were increased indicating impairment of liver functions and generalized tissue damage, respectively, while these increases were significantly reduced by obestatin treatment. Our findings were also confirmed by histological observation. Hepatocyte necrosis areas randomly disseminated in the liver parenchyma with disintegrated cell cordons, leucocyte infiltration, expansion of sinusoids and congestion were displayed after RIR. Treatment with ghrelin markedly reduced these histopathological changes.
In conclusion, since the administration of obestatin inhibited the release of the pro-inflammatory cytokines and the accumulation of neutrophils in the damaged hepatic tissue, these agents appear to play a cytoprotective role in the liver insulted by I/R. It seems likely that obestatin, with its efficiency as antioxidant and antinflammotory, thus merits consideration as a potential therapeutic agent against I/R-induced oxidative remote organ damage.
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