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Thymoquinone protects end organs from abdominal aorta ischemia/reperfusion injury in a rat model

Introduction Previous studies have demonstrated that thymoquinone has protective effects against ischemia reperfusion injury to various organs like lungs, kidneys and liver in different experimental models. Objective We aimed to determine whether thymoquinone has favorable effects on lung, renal, heart tissues and oxidative stress in abdominal aorta ischemia-reperfusion injury. Methods Thirty rats were divided into three groups as sham (n=10), control (n=10) and thymoquinone (TQ) treatment group (n=10). Control and TQ-treatment groups underwent abdominal aorta ischemia for 45 minutes followed by a 120-min period of reperfusion. In the TQ-treatment group, thymoquinone was given 5 minutes. before reperfusion at a dose of 20 mg/kg via an intraperitoneal route. Total antioxidant capacity, total oxidative status (TOS), and oxidative stress index (OSI) in blood serum were measured and lung, kidney, and heart tissue histopathology were evaluated with light microscopy. Results Total oxidative status and oxidative stress index activity in blood samples were statistically higher in the control group compared to the sham and TQ-treatment groups (P<0.001 for TOS and OSI). Control group injury scores were statistically higher compared to sham and TQ-treatment groups (P<0.001 for all comparisons). Conclusion Thymoquinone administered intraperitoneally was effective in reducing oxidative stress and histopathologic injury in an acute abdominal aorta ischemia-reperfusion rat model.


INTRODUCTION
Acute abdominal aorta ischemia followed by reperfusion may be encountered in several clinical circumstances, such as abdominal aortic aneurysm or dissection repair, acute thromboembolism with aortic atherosclerosis, or trauma surgery being brought to the emergency room. Such clinical scenarios are associated with high mortality and morbidity rates due to a systemic inflammatory response and multiple organ dysfunction occurring during the reperfusion phase. Reperfusion of an acutely ischemic aorta may, paradoxically, lead to systemic complications that account for significant morbidity and mortality [1,2] . Overproduction of reactive oxygen species (ROS) and proinflammatory molecules and the subsequent inflammatory response is one of the most crucial underlying mechanisms [2] that initiates injury, especially in the lungs and vital organs, such as kidney and heart, with a subsequent high morbidity [1][2][3][4] .
It has been reported that TQ prevents oxidative injury in various in vitro and in vivo studies [13,14] . TQ possesses strong antioxidant properties through its ability to scavenge different free radicals [15,16] . It has also been reported that TQ attenuated several organ injuries (lung, renal, hepatic) in different ischemia-reperfusion (I/R) models (renal, hepatic). However, no studies have evaluated the protective effects of TQ in an aorta I/R model [17][18][19][20] .
The purpose of this study was to determine the efficacy of TQ in preventing injury in vital organs (lung, heart and kidney) in an acute abdominal aorta ischemia-reperfusion model in rats.

METHODS
The experimental study was performed on a total of 30 three-month-old Wistar-albino rats weighing 200-250 g. All animals were maintained under standard conditions and treated in compliance with National Institutes of Health guidelines. They were housed on a 12-h dark/light cycle schedule with lights on at 06.00 h. Rats were deprived of food, though not water, for 12 hours before surgery. Experiments were done in the Harran University Experimental Research Center. The rats were randomly assigned to three experimental groups: sham operation, control (I/R; non-treated), and TQ-treated I/R. Rats were anesthetized using ketamine hydrochloride (0.2mL/100 g) in all experiments. The abdomen was explored through a midline incision after shaving and disinfection. In the sham group, only laparotomy was performed. In the control group, I/R injury was induced by clamping the aorta under renal vascular pedicles for 45 minutes, followed by 2 hours of reperfusion. In the TQ-treated I/R group, I/R injury was also induced by clamping the aorta under renal vascular pedicles for 45 minutes and TQ was given 5 minutes before reperfusion at a dose of 20 mg/kg via the intraperitoneal route, and again reperfusion was established for 2 hours. Heparin was not used due to possibility of affecting histopathological or biochemical results. At the end of the procedures, the rats were sacrificed after blood sampling, and then kidney, lung, and heart tissues were obtained from all rats.
TQ were purchased from Sigma-Aldrich (St. Louis, MO). The purity (GC) of TQ was ≥98.5% as per the manufacturer's specification and was dissolved in dimethyl sulphoxide.

Measurement of Total Antioxidant Capacity
TAC of supernatant fractions was determined using a novel automated measurement method developed by Erel [21] . do sangue foram medidos, e a histopatologia dos tecidos do pulmão, rim e coração foram avaliados com microscopia de luz.
Hydroxyl radicals, the most potent biological radicals, are produced in this method. In the assay, the ferrous ion solution present in Reagent 1 is mixed with hydrogen peroxide, which is present in Reagent 2. The subsequently produced radicals, such as brown-colored dianisidinyl radical cations produced by the hydroxyl radicals, are also potent radicals. Using this method, the antioxidative effect of the sample was measured against the potent-free radical reactions initiated by the produced hydroxyl radicals. The assay has excellent precision, with values lower than 3%. The results are expressed as nmo-lTrolox Equiv./mg protein.

Measurement of Total Oxidant Status
TOS of supernatant fractions was determined using a novel automated measurement method developed by Erel [22] . Oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundant in the reaction medium. The ferric ion produces a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay was calibrated with hydrogen peroxide, and the results are expressed in terms of nmol H 2 O 2 Equiv/mg protein.

Oxidative Stress Index
The percent ratio of TOS level to TAC level was defined as OSI. OSI values were calculated according to the following formula [23] : OSI (arbitrary unit) = TOS (nmol H 2 O 2 Equiv/mg protein)/TAC (nmolTroloxEquiv/mg protein).

Histopathological Evaluation
The kidney, lung, and heart of each animal were obtained for histological evaluation. Samples of these organs were placed in formalin and embedded in wax according to standard protocols. They were subsequently sectioned at 5 μm slice thickness and stained with hematoxylin and eosin. Magnification of × 20 was used (Olympus BX51 TF, USA). Samples were then graded histologically according to the severity of injury using a predetermined scoring system [24] . The predetermined scoring system, from Solez et al. [24] , included tubular necrosis, interstitial edema, loss of brush border, and cast formation, in which the score was 0 for absent; 1 for mild to moderate; and 2 for marked renal involvement. The histological parameters for lung evaluation were alveolar congestion, intra-alveolar hemorrhage, and interstitial-perivascular infiltration of neutrophils, in which the assessment score was 0 for absent; 1 for mild focal; 2 for moderate focal; and 3 for severe marked lung involvement. Interstitial edema, inflammatory cell infiltration, and coagulation necrosis were assessed for heart examination, in which the score was 0 for absent; 1 for mild focal; 2 for moderate focal; and 3 for severe marked heart involvement. Histological analysis was performed by a blinded expert.

Statistical Analysis
Statistical analyses were performed using SPSS 11.5 (SPSS for Windows 11.5, Chicago, IL). Continuous data are expressed as mean±SD whereas categorical variables are presented as number (count) and percentage. Distribution of continuous variables was assessed with one-sample Kolmogorov-Smirnov test and indicated that all variables were abnormally distributed. Therefore, nonparametric independent group comparisons were made: for multiple comparisons, the Kruskal-Wallis test was used, and for comparisons between groups, the Mann-Whitney test was used if any statistical significance was found. A two-sided P value of <0.05 was considered statistically significant.

RESULTS
All animals survived through the experimental protocol. TAC activity in blood samples were significantly higher in the sham group than in the treatment and control groups  (P<0.001; for all comparisons) but there were no statistically significant differences between the treatment group and control group for TAC activity (P>0.05). TOS and OSI activity in blood samples were statistically higher in the control group than in the sham and thymoquinone group (P<0.001 for all comparisons). Histopathologic injury scores of renal, lung and heart tissues are summarized in Table 1. Control group injury scores were statistically increased compared to sham and thymoquinone groups (P<0.001 for all comparisons). The results are summarized in Figures1, 2, and 3.
Upon histopathological evaluation, renal, lung and heart tissues were found to be normal with no pathological changes in the sham group ( Figures 4A and 4D). Histopathological examination of the tissues in the control group revealed severe lesions, such as tubular damage characterized by cast formation, the loss of brush border and interstitial edema in the kidney. Histopathological examination of the tissues in the control group revealed neutrophil and leukocyte infiltration with alveolar congestion in the lung. Histopathological examination of the tissues in the control group revealed interstitial edema in the heart (Figures 4B and 4E). In rats receiving TQ intraperitoneally, these lesions were less severe than in the control group ( Figures 4C and 4F).

DISCUSSION
In our experimental study, we hypothesized that abdominal aorta ischemia for 45 minutes followed by reperfusion for 2 hours would cause renal, lung, and heart pathology and we have found that (i) abdominal aorta ischemia for 45 minutes followed by reperfusion for 2 hours caused significant pathology in lung, renal, and heart tissues (ii) TOS and OSI levels were increased in the control group and (iii) TOS, OSI, and histopathological injury scores were decreased in sham and TQ+IR groups.
It has been recognized that multiple organ dysfunction syndrome is a major cause of morbidity and mortality after abdominal aortic surgery and contributes to approximately 25% of all deaths in elective abdominal aorta repair. It is postulated that aortic cross-clamping during open repair may cause ischemia-reperfusion (I/R) injury of the intestine and subsequently result in the translocation of bacteria and endotoxin across the intestinal mucosal barrier, leading to the systemic release of reactive oxygen species (ROS) and inflammatory cytokines, which not only damage the gut itself but also harm distant organs, including heart, kidney, and lung [25] .
Nigella sativa (NS), also known as black seed or black cumin, has long been used in folk medicine. NS contains more than 30% of a fixed oil and 0.40-0.45 w/w of a volatile oil. The volatile oil has been shown to contain 18-24% thymoquinone (TQ) and 46% monoterpenes [7] . NS has been reported to exhibit anti-inflammatory, immunomodulatory, and anti-neoplastic effects in many experimental and clinical studies [26][27][28] . TQ, the active constituent of Nigella sativa seeds similar to NS, also showed favorable effects with respect to oxidative stress and inflammation. Thus, TQ has attracted the attention of scientists to investigate its molecular mechanisms and potential use in the treatment of different diseases. It has been shown to have antioxidant/anti-inflammatory effects in several diseases, including experimental allergic encephalomyelitis, colitis, arthritis encephalomyelitis, diabetes, asthma, and carcinogenesis [10] . TQ attenuated lipid peroxidation and increased antioxidant enzyme activities. It has been reported to have strong antioxidant potential through its ability to scavenge different free radicals, its scavenging power being as effective as SOD against superoxide anions [16][17][18] . It acts as a scavenger of superoxide, hydroxyl radicals and singlet molecular oxygen [29] . Furthermore, recent studies have demonstrated that TQ supplementation increases the expression of antioxidant genes, SOD, catalase and glutathione peroxidase in rat liver. Thus, TQ may reduce oxidative stress through a direct antioxidant effect as well as through the induction of endogenous antioxidant enzymes [30] .
TQ also inhibited inducible nitric oxide synthase mRNA expression in rat lipopolysaccharide-stimulated peritoneal macrophage cells [31,32] , which has been attributed to its ability to reduce oxidative stress-induced inflammation leading to the prevention of inducible NOS (nitric oxide synthase) upregulation.
Several studies reported protective effects in the lung in different situations with different mechanisms. Suddek et al. showed that TQ produces a protective mechanism against cysplatin-induced pulmonary damage with anti-oxidant and anti-inflammatory properties and, in addition, TQ has been found to have potential antifibrotic effects besides its antioxidant activity, which could be through NF-κB inhibition, in bleomycin-induced oxidative stress of rat lungs [20,33] . Renal protective effects of TQ have also been discussed in several studies, including vancomycin induced nephrotoxicity, inorganic mercury intoxication, and gentamicin-induced acute renal toxicity. These studies highlight the importance of reactive oxygen species in renal pathophysiology and the intriguing possibility of TQ play a role in the prevention of and/ or protection from renal injury in humans [17,[34][35][36] . Myocardial protective effects of TQ have also been demonstrated in injury induced by isoproterenol, cyclophosphamide-induced cardiotoxicity, and doxorubicin-induced cardiotoxicity [37][38][39] . TQ has also been widely studied in different ischemia reperfusion models and reported to have favorable effects with different potential mechanisms, including primarily antioxidant mechanisms [18,40] . In this study we also found protective effects of TQ in the lung, kidney, and heart with histopathologic evaluation. Significant oxidative stress in the control group compared to sham and TQ groups also emphasizes that the anti-oxidant properties of TQ might be the probable protective mechanism in the acute abdominal aorta ischemia-reperfusion model in the rat.
We believe that there are sufficient preclinical research results with a considerable amount of information about TQ regarding its molecular antioxidant, anti-inflammation, anticancer activity, drug toxicity, bioavailability and pharmacokinetics, and novel drug delivery approaches, to encourage the use of TQ in clinical settings [41] . However, the clinical implications and appropriate pathophysiological mechanisms of the findings of the present study remain to be elucidated with further large-scale clinical studies.
Several limitations of this study should be considered. One of the potential limitations is the absence of oral administration of TQ versus an intraperitoneal route. Another limitation is the absence of biochemical analysis of different biochemical markers, including urea, creatinine, creatinine phosphokinase and creatinine kinase MB for the heart. Further studies focusing on IR injury of other end organs, such as intestine, brain and medulla spinalis injury are needed.

CONCLUSION
In conclusion, TQ administered intraperitoneally was effective in reducing oxidative stress and histopathologic injury in an acute abdominal aorta I/R rat model. Oxidative stress indices and tissue injuries might be modified with TQ treatment in different clinical settings. However, further large scale studies are needed to define the possible favorable effects of TQ in clinical settings.

MSA
Analysis and /or interpretation of data, statistical analysis, final approval of the manuscript conception and study design, conduct of procedures, and/or experiments, writing of the manuscript or review of its content AK Analysis and/or interpretation of data, final approval of manuscript SK Statistical analysis, conception and study design, conduct of procedures, and/or experiments AK Conduct of procedures and/or experiments, writing of the manuscript or review of its content IE Statistical analysis, final approval of manuscript HS Statistical analysis, final approval of the manuscript, conception and study design, conduct of procedures, and/or experiments EB Statistical analysis, final approval of the manuscript, drafting the manuscript or revising it critically for its content AH Drafting of the manuscript or review of its content