Effects of iloprost and n-acetylcysteine on ischemia-reperfusion injury and thiol/disulfide hemostasis in rats

Abstract

AimIntestinal ischemia occurs after partial or complete obstruction of the intestinal arterial blood flow, and reperfusion injury following the restoration of blood flow. Intestinal ischemia-reperfusion [IIR] damage can cause multiple organ failure and death.
In our study, we aimed to observe the effect of ilioprost and N-acetylcysteine on ischemia-reperfusion injury and to show the effect of the Thiol disulfide mechanism in this area.MethodsThirty Sprague Dawley rats were divided into five groups of six animals each: sham, IIR, IIR+IL, IIR+NAC and IIR+NAC+IL. Intestinal samples and blood were collected after completion of the sham or IIR protocol. Small-bowel samples were evaluated according to the Chiu score. Thiol/disulfide [DS] hemostasis was followed using a novel series of serum biomarkers. Serum concentrations of total thiol, native thiol and disulfide were also determined.ResultsThe average Chiu score was lower in the IIR + NAC group than in both the IIR and the IIR + IL group, but the differences were not statistically significant. The score in the sham group was significantly lower than those of the other four groups. The level of reduced thiol and the native thiol/total thiol [NT/TT] ratios were higher in groups treated with NAC, IL or both. In the latter groups, oxidized thiol, DS/TT and DS/NT ratios were lower than in the IIR group but the differences between the three treatment groups were not statistically significant.ConclusionThe addition of IL to NAC was not more protective than NAC alone in a rat model of IIR injury. Our results suggest that markers of thiol-DS hemostasis can be used as indicators of antioxidant mechanisms in IIR injury.

Keywords

Introduction

Ischaemia is defined as reversible or irreversible cell or tissue death due to the depletion of cell energy stores and the accumulation of toxic substances. This is the result of an insufficient blood supply to the tissues and organs due to decreased arterial and/or venous blood flow. Ischaemia causes tissue hypoxia and thus hypoxic tissue damage.¹ However, the restoration of blood flow.[reperfusion] induces even more damage.² including the formation of reactive oxygen species (ROS) from the renewed supply of cellular oxygen.³ Mesenteric ischaemia that develops due to the occlusion of the superior mesenteric artery (SMA) prominently affects the small intestine, which is highly susceptible to ischemia-reperfusion (IR) injury.^4,5 Iloprost (IL) is a stable prostaglandin I2 analog with pharmacokinetic properties very similar to those of the parent compound.^6,7 Previous studies have demonstrated the protective effects of IL on IR injury in different organs.⁸ N-acetylcysteine (NAC) is an intracellular glutathione (GSH) precursor that increases glutathione S-transferase activity in the liver.⁹ Its activities include the protection of tissues against ROS damage, by directly removing oxidants.^10,11 The beneficial effects of NAC in rats, including rat models of IR injury of the heart and liver, have been demonstrated.^11,12 Thiols (-SH) are organic compounds that contain a sulfhydryl functional group.¹³ Among the thiols found in plasma are albumin and other proteins, cysteine, cysteinyl glycine, glutathione, homocysteine and γ-glutamyl cysteine thiols.¹⁴ Oxidation of the thiol groups of these thiol-containing molecules results in the formation of reversible disulfide (DS) bonds.¹⁵ The reversible oxidation-reduction of the thiol groups and DS bonds is referred to as thiol-DS hemostasis.¹⁶ and it can be followed using the method developed by Erel and Neşelioğlu.¹⁷ which allows each and/or all of the components to be measured and further analyzed. In this study, we used of this method to investigate the abilities of IL and NAC, alone or in combination, to prevent intestinal IR (IIR) injury in rats.

Materials and Methods

Experimental DesignEthical approval was obtained from the Local Ethics Committee on Animal Experiments of Sakarya University University on 05/09/2018 with decision No. 24. Thirty mixed-sex Sprague Dawley rats weighing 250–350 g each were divided into five groups of six animals: sham, IIR, IIR+IL, IIR+NAC and IIR+NAC +IL. Prior to the experiment, the rats were acclimated under 12 h light/12 h dark conditions, with water and standard rat food supplied ad libitum. Intestinal samples.[5 cm of ileum from the distal end of the terminal ileum to the proximal ileum] and blood were collected from the sham group, which did not undergo any surgical procedures. In the IIR group, the cranial mesenteric artery.[CMA] and the superior mesenteric artery (SMA) were dissected and then clamped to induce intestinal ischemia. Then the skin was closed with skin staples. After 45 min of ischemia, the abdomen of the rats was reoperated and reperfusion was induced by declamping the CMA with the skin again closed using skin staples. After 120 min of reperfusion, the abdomen was reoperated and the animals were euthanized by cervical dislocation. Intestinal samples and blood were collected. In the IL-treated rats, 2 µg IL/kg was administered intraperitoneally 30 min before reperfusion. In the NAC-treated rats, 300 mg NAC/kg was administered intraperitoneally 30 min before reperfusion. Intestinal samples and blood were collected as in the sham group.Histopathological EvaluationTissues separated for histopathological examination were fixed in 10% formaldehyde and sent to the Department of Pathology. After routine processing, the tissue samples were embedded in paraffin, from which sections 5 µm thick were cut and stained with hematoxylin and eosin. Sections were evaluated by light microscopy (Nikon Eclipse-Ni Y-THPL, Japan) by a pathologist blinded to the treatment groups. The tissues were classified using the system of Chiu et al. (1970), described in Table1.Biochemical EvaluationThiol-DS Hemostasis MeasurementsThe concentrations of total thiol (TT, μmol/L) and native thiol (NT, μmol/L) in serum were measured and the values were used to calculate the thiol/DS ratio, an oxidative marker of hemostasis. Samples collected in capped gel tubes and transferred in a cold chain were allowed to stand for 30 min before they were centrifuged (cooled, 4000 rpm 10 min). Serum parameters of interest were analyzed using Rel assay diagnostics kits. A fully automated autoanalyzer (Beckman Coulter, chemistry analyzer AU 680, serial number: 2016024580, MishIMA K.K, Japan) in the Biochemistry Laboratory was used to measure TT, NT and DS concentrations (μmol/L), from which DS/NT, DS/TT and NT/TT ratios, as well as oxidation reduction rates, were determined.Ethical ApprovalEthics Committe approval for the study was obtained.Statistical AnalysesAll data were processed using IBM Statistical Package for the Social Sciences Statistics 22 (SPSS 2013). Numerical variables were analyzed using descriptive statistics.[mean, standard deviation], and differences between categorical variables from more than two groups were analyzed via one-way ANOVA. A Levene test was performed to determine the presence or absence of variance homogeneity, followed by a Tukey test or Tamhane’s T2 test.

Results

Histopathological ResultsA comparison of the Chiu scores of the five groups (Table 2) showed that they were lower in the sham group than in all other groups. Microscopic examination of the small intestine sections of the sham group showed that all were of grade 0. Thus, there were no morphological changes in the lamina propria, and ulceration, mononuclear cell infiltration, increased capillary permeability and hemorrhage were not observed (Figure 1a). In sections of small intestine with grade 1 damage (IIR+NAC and IIR+NAC+IL), subepithelial separations were present at the upper end of the villi (Figure 1b). In rats with grade 2 damage, the subepithelial separations were of moderate grade (Figure 1c). Grade 3 damage, seen in all IIR groups, consisted of deformations at the ends of the villi where the mucosal epithelium was pushed intensely upwards (Figure 1d). In rats with grade 4 damage (IIR, IIR+IL, IIR+NAC+IL), villus deformation was accompanied by dilated capillaries reaching the lamina propria (Figure 1e).
IIR scores were lower in the IIR + IL group than in the IIR + NAC and IIR + NAC + IL groups, but the differences were not statistically significant (P>0.05). All IL groups had a higher mean Chiu score than the IIR group, but again the difference was not significant. The IIR + NAC group had a lower Chiu score than either the IIR group or the IIR + IL group, but neither difference was statistically significant (Figure 2). The Chiu score of the IIR + NAC + IL group was lower than that of the IIR and IIR + IL groups and higher than that of the IIR + NAC group, without statistically significant differences. The mean Chiu score of the IIR + NAC + IL group was significantly higher than that of the sham group.Thiol-Disulfide HemostasisThe results of biochemical analyses of thiol-DS hemostasis in the five groups of rats are presented in Table 3.
According to the results of one-way ANOVA, TT, NT, DS, NT/TT, DS/TT, DS/NT, the reduced thiol ratio, the oxidized thiol ratio and the thiol oxidation reduction rates differed significantly among the five groups (P<0.05). The data were further analyzed in a Levene test followed by a Tukey test for homogeneous groups and a Tamhane T2 test for non-homogeneous groups. TT levels (Figure 3) were lower in the IIR and IIR + IL groups than in the sham group, but without a significant difference (P>0.05). However, they were significantly higher in the IIR + NAC and IRR + NAC + IL groups than in the other groups and in the IIR + NAC group than in either the IIR + IL or the IIR + NAC + IL group.
Similar to TT, NT levels (Figure 3) were lower in the IIR and IIR + IL groups than in the sham group but the difference was not significant (P>0.05). The levels were significantly higher in the IIR + NAC and IIR + NAC + IL groups than in the sham and IIR groups, but significantly lower in the IIR + IL group than in the IIR + NAC group.
DS levels (Figure 3) were significantly higher in the sham, IIR + NAC and IIR + NAC + IL groups than in the IIR + IL group (P<0.05), which was slightly higher than in the IIR group. The difference between the IIR + NAC + IL group and the IIR group was significant. The reduced thiol ratio was significantly higher in the IIR + IL, IIR + NAC, and IIR + NAC + IL groups than in the sham and IIR groups (P<0.05). It was higher, but not significantly, in the IIR + NAC group than in the IIR + NAC + IL and IIR + IL groups. The NT/TT ratio was significantly higher in the IIR + IL, IIR + NAC, IIR + NAC + IL groups than in the sham and IIR groups and was also higher in the IIR + NAC group than in the IIR + NAC + IL and IIR + IL groups, but the differences were not significant. The oxidised thiol ratio was significantly lower in the IIR + IL, IIR + NAC, IIR + NAC + IL groups than in the sham and IIR groups (P<0.05) but the difference between the IIR + NAC + IL and IIR + IL groups was not statistically significant. The DS/NT ratio was significantly lower in the IIR + NAC, IIR + IL and IIR + NAC + IL groups than in the IIR group; significantly higher in the IIR + NAC group than in the sham group; and non-significantly lower in the IIR + NAC group than in both the IIR + NAC + IL and IIR + IL group. The DS/TT ratio was significantly lower in the IIR + IL, IIR + NAC, IIR + NAC + IL groups than in the sham and IIR groups and non-significantly lower in the IIR + NAC group than in the IIR + NAC + IL and IIR + IL groups. The thiol oxidation reduction rate was significantly higher in the IIR + NAC + IL group than in the IIR group, and non-significantly higher in the sham and all treated groups than in the IIR group.

Discussion

Our study uniquely evaluated thiol-DS hemostasis in a rat model of IIR injury. Although methods to reduce IR injury and prevent organ failure have been described in the literature.¹⁹ our study is the first to compare the effects of IL and NAC on reperfusion subsequent to intestinal ischemia.
The protective effects of IL on ischemic musculoskeletal damage and in heart disease, lung diseases, and IR-associated spinal cord injury have been demonstrated in several studies.^20-25 Using the Chiu score, we found that IL reduced IR injury in the small intestine compared to the untreated (IR) control, but the difference was not statistically significant.
However, our study is the first to investigate the effects of these two drugs on intestinal IR. A comparison of IL and NAC, alone or in combination, showed better histopathological results in NAC-treated groups and worse histopathological results in IL groups. This finding supports previous studies that have reported that NAC may be effective for the prevention of IIR injury. Thus, the therapeutic effects of NAC in the clinical setting merit investigation.
Whether IL and/or NAC, as antioxidants, have an effect on thiol-DS hemostasis, a new marker of oxidative stress was also examined in this study. While many clinical conditions disrupt the thiol-DS balance, it is not known whether this also occurs in IIR injury. Erel and Neselioğlu.¹⁷ showed that plasma levels of DS were higher in patients with degenerative diseases such as diabetes, obesity and pneumonia as well as in smokers, but lower in patients with proliferative diseases such as multiple myeloma, bladder cancer, colon cancer and kidney cancer
Our study revealed significant differences between groups with respect to serum markers for thiol-DS haemostasis. Specifically, TT, NT and DS levels were highest in the NAC group and lower in the NAC+IL groups. The oxidised thiol level was the lowest and the reduced thiol ratio was the highest in the NAC group, which suggests that IL reduces the effects of NAC when the two drugs are administered in combination. Our results demonstrate that utility of TT, NT and oxidized and reduced thiol ratios in assessing the degree of antioxidant activity during IIR injury. This novel finding should be examined in further studies. The limitation of this study is that the number of subjects in the study was small.

Conclusion

A combination of NAC and IL has no advantage over NAC alone in protector IIR injury. The use of the Thiol disulfide mechanism and measurement parameters in intestinal ischemia-reperfusion injury gives results related to the rate of damage. New studies are needed to increase the level of evidence of the results.

Declarations

References

1. Ozan E, Koyutürk L, Sapmaz T. Böbrek iskemi-reperfüzyon hasarında antioksidan olarak prostaglandin E1 (PGE1) kullanımının incelenmesi: deneysel çalışma [Investigation of the use of prostaglandin E1 (PGE1) as an antioxidant in kidney ischemia-reperfusion injury: an experimental study]. Fırat Tıp Derg. 2004;9(3):67-71.
   PubMed · Google Scholar
2. Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention of ischemia-reperfusion injury. Anesthesiology. 2001;94(6):1133-1138. doi:10.1097/00000542-200106000-00030
   Article · PubMed · Google Scholar
3. Wilhelm J. Metabolic aspects of membrane lipid peroxidation. Acta Univ Carol Med Monogr. 1990;137:1-53.
   PubMed · Google Scholar
4. Güneli E, Çavdar Z, İşlekel H, et al. Erythropoietin protects the intestine against ischemia/reperfusion injury in rats. Mol Med. 2007;13(9-10):509-517. doi:10.2119/2007-00032.guneli
   Article · PubMed · Google Scholar
5. Rock P, Yao Z. Ischemia reperfusion injury, preconditioning, and critical illness. Curr Opin Anaesthesiol. 2002;15(2):139-146. doi:10.1097/00001503-200204000-00001
   Article · PubMed · Google Scholar
6. Ylitalo P, Kaukinen S, Seppälä E, et al. Pharmacological effects of iloprost (ZK 36 374), a stable prostacyclin analogue, in man. Biomed Biochim Acta. 1984;43(8-9):399-402. doi:10.1038/clpt.1984.205
   Article · PubMed · Google Scholar
7. Stürzebecher CS, Losert W. Effects of iloprost on platelet activation in vitro. In: Gryglewski RJ, Stock G, eds. Prostacyclin and Its Stable Analogue Iloprost. Springer; 1987:39-45. doi:10.1007/978-3-642-71499-3_4
   Article · PubMed · Google Scholar
8. Avcı T, Erer D, Kucuk A. The effects of iloprost on ischemia-reperfusion injury in skeletal muscles in a rodent model. J Surg Res. 2014;187(1):162-168.
   PubMed · Google Scholar
9. Slusser SO, Grotyohann LW, Martin LF, Scaduto RC Jr. Glutathione catabolism by the ischemic rat kidney. Am J Physiol. 1990;258(6). doi:10.1152/ajprenal.1990.258.6.f1547
   Article · PubMed · Google Scholar
10. Conesa EL, Valero F, Nadal JC, et al. N-acetyl-L-cysteine improves renal medullary hypoperfusion in acute renal failure. Am J Physiol Regul Integr Comp Physiol. 2001;281(3). doi:10.1152/ajpregu.2001.281.3.r730
    Article · PubMed · Google Scholar
11. Abbasoglu SD, Balkan J, Kanbagli O, et al. Aminoguanidine, an inducible nitric oxide synthase inhibitor, plus N-acetylcysteine treatment reduce the lipopolysaccharide-augmented hepatotoxicity in rats with cirrhosis. Hum Exp Toxicol. 2002;21(7):359-364.
    PubMed · Google Scholar
12. Bartekova M, Barancik M, Ferenczyova K, Dhalla NS. Beneficial effects of N-acetylcysteine and N-mercaptopropionylglycine on ischemia reperfusion injury in the heart. Curr Med Chem. 2018;25(3):355-366. doi:10.2174/0929867324666170608111917
    Article · PubMed · Google Scholar
13. Sen CK, Packer L. Thiol homeostasis and supplements in physical exercise. Am J Clin Nutr. 2000;72(2 suppl):653S-669S. doi:10.1093/ajcn/72.2.653s
    Article · PubMed · Google Scholar
14. Turell L, Radi R, Alvarez B. The thiol pool in human plasma: the central contribution of albumin to redox processes. Free Radic Biol Med. 2013;65:244-253. doi:10.1016/j.freeradbiomed.2013.05.050
    Article · PubMed · Google Scholar
15. Cremers CM, Jakob U. Oxidant sensing by reversible disulfide bond formation. J Biol Chem. 2013;288(37):26489-26496. doi:10.1074/jbc.r113.462929
    Article · PubMed · Google Scholar
16. Jones DP, Liang Y. Measuring the poise of thiol/disulfide couples in vivo. Free Radic Biol Med. 2009;47(10):1329-1338. doi:10.1016/j.freeradbiomed.2009.08.021
    Article · PubMed · Google Scholar
17. Erel O, Neselioglu S. A novel and automated assay for thiol/disulphide homeostasis. Clin Biochem. 2014;47:326-332. doi:10.1016/j.clinbiochem.2014.09.026
    Article · PubMed · Google Scholar
18. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-flow states: a morphological, hemodynamic, and metabolic reappraisal. Arch Surg. 1970;101(4):478-483. doi:10.1001/archsurg.1970.01340280030009
    Article · PubMed · Google Scholar
19. Eriş O, Çankayalı İ, Sezer E. Ratlarda oluşturulan hemorajik şok modelinde N-asetilsisteinin oksidatif stres üzerine olan etkisi [The effect of N-acetylcysteine on oxidative stress in the hemorrhagic shock model created in rats]. Turk J Anaesthesiol Reanim. 2009;37(4):208-216.
    PubMed · Google Scholar
20. Caliskan A, Yavuz C, Karahan O, et al. Iloprost reduces myocardial edema in a rat model of myocardial ischemia reperfusion. Perfusion. 2014;29(3):260-264. doi:10.1177/0267659113514472
    Article · PubMed · Google Scholar
21. Sacar M, Ozcan V, Aybek H, et al. Vitamin C and iloprost attenuate skeletal muscle injury caused by ischemia-reperfusion of the lower extremities. Turk Gogus Kalp Damar Cerrahisi Derg. 2005;13(4):374-378.
    PubMed · Google Scholar
22. Koksel O, Ozdulger A, Aytacoglu B, et al. The influence of iloprost on acute lung injury induced by hind limb ischemia-reperfusion in rats. Pulm Pharmacol Ther. 2005;18(4):235-241. doi:10.1016/j.pupt.2004.12.005
    Article · PubMed · Google Scholar
23. Katircioglu SF, Gokce P, Ozgencil E, et al. Prostacyclin usage for spinal cord protection during experimental thoracic aortic cross-clamping. Vasc Endovascular Surg. 1996;30(2):97-101.
    PubMed · Google Scholar
24. Aytacoglu BN, Sucu N, Tamer L, et al. Iloprost for the attenuation of ischaemia/reperfusion injury in a distant organ. Cell Biochem Funct. 2006;24(4):341-346. doi:10.1002/cbf.1223
    Article · PubMed · Google Scholar
25. Yakut N, Yasa H, Bahriye LB, et al. The influence of levosimendan and iloprost on renal ischemia-reperfusion: an experimental study. Interact Cardiovasc Thorac Surg. 2008;7(2):235-239. doi:10.1510/icvts.2007.161356
    Article · PubMed · Google Scholar

Tables