Abstract

Objective

The aim of the present study was to investigate the effect of thymoquinone on ischemia/reperfusion (I/R) injury at 150 min or/and 24 h of reperfusion in male Wistar Rats.

Material and Methods

The therapeutic value of thymoquinone on cellular damage caused by reactive oxygene species or inflammatory processes during intestinal ischemia/reperfusion was investigated using pharmacological function studies on smooth muscle contractile responses of acetylcholine (Ach) and KCl, along with myeloperoxidase activity, malondialdehyhde, glutathione and cytokine levels such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β in serum and ileum tissue of rats. Thymoquinone was administered at a dose of 50 mg/kg orally for three times: 30 min, 24 h and 48 h prior to the surgical procedure. Soon after reperfusion timing (150 min or 24 h), the contractility traces to KCl and acetylcholine of the ileum smooth muscle were recorded through isolated organ bath.

Results

Pretreatment with thymoquinone reversed the disrupted contractility of the ileum smooth muscle at the 24 h reperfusion. Increased malondialdehyde and depleted glutathione levels and high myeloperoxidase activity determined in the ileum I/R tissue returned to reasonable amounts by pretreatment of Thymoquinone, which attenuated malondialdehyde quantity, restored glutathione level and inhibited myeloperoxidase activity. In addition, both serum and tissue TNF-α and IL-1β activities were modulated by thymoquinone at 24 h of intestinal I/R.

Conclusion

The results indicate that thymoquinone may have therapeutic value due to its immunomodulating, radical scavenging and/or antioxidant effects in intestinal I/R injury including oxidant damage mechanisms.

Introduction

Ischemia due to insufficient blood supply to tissues results in cellular function failure; however, reperfusion exacerbates the ischemic damage more. Actually, the intestinal tissue is quite sensitive to ischemia/reperfusion (I/R) through mesenteric artery. Cellular damages during surgical procedures or pathological conditions including bowel transplantation, acute mesenteric ischemia, abdominal aortic aneurysm, and shock are remarked as the main causes of intestinal I/R injury. Increased protein extravasations, disruption of mucosal barrier, decreased contractile activity, and impairment of gut motility are clearly observed with intestinal I/R (1). The activation of inflammatory cells such as polymorphonuclear leukocytes leads to fast exagreation of the onset of inflammatory reaction. Previous studies have shown that in the pathologenesis of I/R, there is an increase in the amounts of reactive oxygen and nitrogen species, cytokines, endotoxins, and neutrophils (2-4). Several studies have also demonstrated that neutrophils, adhesive molecules and endothelial cells are responsible for serious and deleterious cellular inflammatory dysfunction caused by intestinal I/R injury (3, 4). It is well known that the produced reactive oxygen or nitrogen species and oxidative damage are the main responsibles of cell death and organ dysfunction in intestinal I/R (3, 5).

Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone) is a therapeutically active chemical structure of the essential and fixed oil obtained from Nigella sativa seeds. Recently, data have addressed on its in vivo and in vitro pharmacotherapeutic effects (6). Its antioxidant, anti-inflammatory or immunmodulatory actions have been revealed in several disease models (7-11), as well as in gastric ulcer caused by I/R (9). Moreover, TQ has been shown to inhibit the accumulation of inflammatory cells in bronchial alveolar fluid (BALF) and lung tissue (12). In a study of Tas et al., TQ has been found to cause decreased levels of malondialdehyde (MDA) in ischemia-reperfusion injury while increasing the level of glutathione (GSH) (13). A recent study reveals that TQ reverses cytokine increases such as tumor necrosis factor (TNF)-α and interleukin (IL)s caused by I/R in both serum and intestinal tissue (13).

For all reasons, it is to prove the hypothesis that the antioxidant and antiinflammatory role of TQ in I/R injury will reduce injury of intestinal ischemia reperfusion. Therefore, this research was planned to reveal the effect of TQ on intestinal I/R.

Material and Methods

Animals

This study was conducted with forty-eight male Wistar-albino rats weighing 200-250 g in Experimental Research Section of Duzce University. The approval of the animal ethical committee of Duzce University was taken with the number: 2011/009.

Thymoquinone Treatment

All chemicals used in the study was obtained from Sigma (USA) and was prepared with 1% Tween 80 to be administered orally. TQ (50 mg/kg of body weight per day) or gavage canula (1 mL) was given orally once a day for 3 days before surgery and 30 min before surgical procedures (n= 8). The dose of TQ was administered as done by El-Abhar et al. in their study (14).

Experimental Protocols of Induction of Ischemia/Reperfusion in Intestinal Tissue

Intestinal I/R was performed as described previously (3). Briefly, under sodium thiopental anaesthetize midline laparotomy was performed, the small intestine was gently naked with humid sterile gauze to block dehydration. After I/R was performed through the occlusion of superior mesenteric artery with a 30-minute schedule using microvascular clamp, ileum was perfused by 150 min. Loss of pulsation and coloration in the bowel was observed in the ischemia period. To let blood flow to the intestines, the clamp was opened gently at the end of 30 minutes. In the experimental protocol, groups were divided as follows:

I. Sham group (n= 16): The rats underwent laparotomy, without ischemia/reperfusion, remained open until the ischemia/reperfusion period or after 30 minutes of follow-up, the abdominal wall was sutured.

II. I/R-vehicle group without any treated (n= 16): First, the superior mesenteric artery was occluded with clamp for 30 min and then ischemia was started. Immediately 150 minutes or 24 h reperfusion was performed and this procedure was also done for the I/R-vehicle after vehicle was given.

III. I/R and TQ-treated group (n= 16): Animals subjected to ischemia/reperfusion were preadministered with TQ as described above.

Animals were divided into 2 series for 150 min and 24 h reperfusion studies (n= 8). Bioochemical analyses were only measured at 24 h.

Preparation of the Terminal Ileum and Contractile Studies in Intestinal Tissue

The aim of this process was to evaluate the contractile activity of the ileal longitudinal muscle in isolated ileal segments in organ bath (Commant Iletisim Co, Ankara, Turkey). A 15-mm length terminal ileum tissue fragment was cleaned and immediately suspended in an isolated organ bath with a Krebs solution involving KCl 4.7, NaHCO3 24.88, MgSO4 1.16, KH2PO4 1.18, CaCl2 2.52, NaCl 118, and glucose 11.1 in mM. Longitudinal segments of the ileum smooth muscle were calibrated during 60 min at 2 g force in organ bath filled with Krebs solution regularly fed with a gas mixture of 5% CO2 and 95% O2 at 37°C. Following the 2 g tension equilibration, spontaneous activity, 30 mM KCl, and cumulative acetylcholine (Ach) contractility were recorded.

In order to measure cytokines, myeloperoxidase (MPO), glutathione (GSH), and malondialdehyde (MDA) activity, harvested blood and then tissues were immediately removed and cleaned with buffer solution and stored at -80°C deep freeze.

MDA Determination in the Intestinal Tissue

MDA levels were detected using a method described by Casini et al. based on thiobarbituric acid reaction (15).

Determine of GSH Levels in Intestinal Tissue

The method described by Ellman was used to determine the level of GSH in intestinal tissue (16).

Determinationt of MPO Activity in Ileum Homogenate

The method described by Bradley et al. was used to determine the amount of myeloperoxidase (MPO), which is an indicator of migration of the neutrophils to the inflammed tissue (17).

Determination of TNF-α and IL-1β Activities in Serum and Intestinal Tissue

In both serum and tissue, the activity of TNF-α and IL-1β was measured by following the instructions in the manufacturer’s guideline papers (abcam ab100770, ab100768, respectively, Istanbul/Turkey).

Statistical Analysis

Statistical analysis was performed using a Kruskal-Wallis test, followed by a post hoc Bonferroni test to estimate the differences between groups. A two-way ANOVA with multiple post hoc comparisons performed with the Bonferroni test was used to determine the differences between the 150 min and 24 h series.

Results

Ileal Longitudinal Muscle Contractility

As seen in Figure 1 with original traces, the cumulative dose of Ach (10-8-10-3 M) produced concentration-dependent contraction on isolated ileum in the sham, I/R-vehicle and TQ at 24 h after ischemia procedure. Contraction response of the cumulative dosing of Ach in the TQ group was almost as much as the response of the sham group at 24 h after ischemia while the cumulative effect of Ach in the 150-minute reperfusion group was not statistically significant. In 150 min reperfusion periods, statistical significance was not significant in the 150-minute reperfusion pediods as shown in Table 1 and Figure 2.

There were statistical differences between I/R-vehicle and sham groups in the Ach contraction responses both at 150 min (Figure 2A) and 24 h (Figure 2B) of the reperfusion periods. The inhibition of Ach-induced contraction due to I/R was reversed by TQ for the responses to 10-6 M-10-3 M Ach at 24 h (p< 0.05, p< 0.0001), however, this effect of TQ was not observed at 150 min of reperfusion (Table 1). Therefore, biochemical analyses were only measured at 24 h.

At 24 h of reperfusion, the depressed response of KCl-induced contractions in the TQ treated I/R-vehicle group was reversed and a similarity was seen in the sham control group (Figure 3). The ameliorating affects of TQ on the Ach and KCl contractile responses of the intestinal I/R tissue were observed only at 24 h of reperfusion even though there was no gain at 150 min of reperfusion (Table 1). Moreover, there was a statistical difference between the 150 min and 24 h reperfusion periods at 30 mM KCl when comparing TQ groups with each other.

MDA Levels in the Intestinal Tissue at the 24 h of Reperfusion

As shown in Figure 4A and Table 2, in the I/R-vehicle group, MDA level in the ileum homogenate was observed significantly higher than the Sham group at 24 h of reperfusion. Pre-treatment of a 50 mg/kg dose of TQ significantly inhibited out the content of MDA.

GSH Levels in the Intestinal Tissue at the 24 h of Reperfusion

As illustrated in Figure 4B and Table 2, GSH levels in the intestinal homogenate of the I/R-vehicle animals were observed significantly lower than the sham group at 24 h of reperfusion. Pre-treatment with a 50 mg/kg dose of TQ, however, significantly improved the decreased quantity of GSH in theTQ pre-administered I/R-vehicle group, with a significantly higher GSH level.

MPO Activity in the Intestinal Tissue at the 24 h of Reperfusion

Figure 4C shows that the statistical significances were found between I/R-vehicle and sham groups and between TQ and I/R-vehicle groups at 24 h of reperfusion (Table 2).

Level of TNF-α in Serum and Intestinal Tissue at the 24 h of Reperfusion

As shown in Figure 5A and 5B, in the I/R-vehicle group, the TNF-α levels in serum and ileum homogenate were observed significantly higher than the Sham group at 24 h of reperfusion. Pre-treatment of a 50 mg/kg dose of TQ significantly inhibited out the levels of TNF-α in the serum and tissue homogenate.

IL-1β Assay in Serum and Intestinal Tissue at the 24 h of Reperfusion

The other criteria of the size of the I/R-induced ileum injury is the determination of IL-1β levels in the serum and ileum tissue. As illustrated in Table 2, when the I/R-vehicle groups and sham groups were compared in terms of IL-1β levels in both the serum and ileum homogenate, the increase in all of these parameters in the I/R-vehicle groups was statistically different. There was a statistically significant difference when compared to a 50 mg/kg dose of TQ group with I/R-vehicle group in the serum (Figure 5C) and tissue homogenate (Figure 5D).

Discussion

The results of the present research revealed that intestinal I/R resulted in a depressed ileum contractile responses to KCl, unspecific K ion channels related to contractility, and Ach, an excitatory neurotransmitter that effects by cholinergic induction in the smooth muscle cells of the digestive system. TQ administration was noticed ameliorate the distrupted contractile responses at 24 h after I/R, whereas it seemed to have no improvement at 150 min of I/R.

The ileum contraction response due to I/R damage of intestinal tissue is significantly impaired (3). Inflammatory mediators, ROS, leukocyte migration, and events in the immune processes can trigger the contractile chaos of the smooth muscle induced by I/R. It is known that I/R causes endothelium integrity, endothelial dysfunction, and failure in the oxydant defence mechanisms (2, 3, 5). In order to understand the therapeutic effect of TQ in intestinal I/R injury well, markers of oxidative stress and inflammation such as MDA, GSH, MPO and TNF-α activities in the ileum tissue were investigated. The results indicated that stimulated cytokine, neutrophilic inflammation and oxidative events play a role in I/R-induced intestinal damage. Fortunately, pretreatment of animals with TQ restored intestinal dysfunction, reduced elevated MDA, MPO, TNF-α levels and reversed the depleted intestine GSH levels at I/R 24.

One of the unpredictive result was that we did not observe the reverse effect of distrupted contractily at the 150th min of reperfusion, while it was significantly decreased at the 24 h of reperfusion by TQ administration. Actually, a dose of 50 mg/kg/bw of TQ was chosen according to a previous paper (13, 14). That dose may be insufficient to carry out a therapy for acute I/R injury. Another limitation about our research protocol may be the oral route of application of TQ to animals. In several investigations, unlike us, the application of antioxidant agent has been intravenous infusion just before ischemia (5). On the other hand, active TQ in the body can affect the liver due to its inactive biotransformation tract (18). Therefore, future studies are needed to plan the application routes of TQ in pathogenesis of I/R injury in detail.

The results of the present study on smooth muscle functions revealed that intestinal I/R injury was appreciably reduced by TQ preapplication. According to the literature, oxidant stress is a basic mechanism on inflammation and pathogenesis in intestinal I/R injury (13). This study revealed that increased MDA levels reversed with three times TQ administration prior to I/R surgery operation. In addition, GSH has non-enzymatic antioxidant structures of cells to protect against to oxidant processes (13, 19). TQ elevated the deppressed GSH level of intestinal cells. These results indicate that TQ has attributed to the upregulation of endogenous cellular antioxidant systems during the progress of subacute I/R injury. MPO activity is often discussed to show the extent of inflammation in intestinal tissues subjected to I/R injury. Our study revealed that the increased MPO activity inhibited with three times TQ administration prior to I/R surgery operation.

Recent studies have revealed that the suppression of overproduction of pro-inflammatory cytokines including TNF-α and IL-1β occurs in exaggerated immunity or inflammation of disease models such as asthma, rheumatic arthritis, cancers, neurodegenerative diseases, cardiotoxicity, and etc. by TQ (12, 20-24). Our results showed that TQ administration inhibits both ileum tissue and plasma TNF-α and IL-1β expressions in intestinal I/R injury. Cytokine production ring can occur due to the migration of polymorphonuclear leukocytes to the injured tissue.

Some limitations of this study are about other probable effect mechanisms of TQ on I/R injured tissue pathogenesis. One mechanism of TQ may be that it enhances the expression of endothelial nitric oxide synthase and increases nitric oxide levels, potent antioxidant which reacts with toxic molecular radicals, along with down-regulated nitric oxide synthase expression (25). TQ may have improved effects on endoplasmic stress and mitochondrial dysfunction and anti-apoptotic effects through activation of autophagy in damaged cells following numerous I/R models (13, 26-29).

Conclusion

Our results confirm that TQ has antioxidant and aniinflammatory activities in the prevention and therapy of intestinal I/R injury. However, for clinical use, the dose, effect, and safety of TQ must be investigated by clinical phase studies in healthy volunteers or patients with numerous diseases. Finally, the results of the current study clearly reveal the role of oxidative damage in the immuno-pathophysiology of intestinal I/R injury, and in fact, TQ can be useful as a prophylactic and therapeutic agent in intestinal I/R injury.