INTRODUCTION

---

The lazaroid U-74389G (L) may be not famous for its hypertransferasemic[1] capacity (P = 0.0583). U-74389G as a novel antioxidant factor implicates exactly only 260 published studies. The ischemiareperfusion (IR) type of experiments was noted in 18.84% of these studies. A tissue protective feature of U-74389G was obvious in these IR studies. The U-74389G chemically known as 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1- piperazinyl]-pregna-1,4,9(11)-triene-3,20-dione maleate salt is an antioxidant complex, which prevents the lipid peroxidation either iron-dependent or arachidonic acidinduced one. Animals' kidney, liver, brain microvascular endothelial cell monolayers, and heart models were protected by U-74389G after IR injury. U-74389G also attenuates the leukocytes, downregulates the pro-inflammatory gene, treats the endotoxin shock, produces cytokine, enhances the mononuclear immunity, protects the endothelium, and presents anti-shock property.

Erythropoietin (Epo) even if is not famous for its hypertransferasemic action (P = 0.4430), it can be used as a reference drug for comparison with U-74389G. Although Epo is met in over 30,606 published biomedical studies, only a 3.57% of them negotiate the known type of IR experiments. Nevertheless, Epo as a cytokine, it is worth of being studied about its effects on aspartate aminotransferase levels (AST) levels too.

This experimental work tried to compare the effects of the above drugs on a rat-induced IR protocol. They were tested by calculating the serum AST level (sASTl) augmentations.

MATERIALS AND METHODS

---

Animal preparation
The Vet licenses under 3693/12-11- 2010 and 14/10-1-2012 numbers, the granting company, and the experiment location are mentioned in preliminary references[1,2]. The human animal care of albino female Wistar rats, 7 days' pre-experimental ad libitum diet, non-stop intraexperimental anesthesiologic techniques, acidometry, electrocardiogram, oxygen supply, and post-experimental euthanasia are also described in preliminary references. Rats were 16-18 weeks old. They were randomly assigned to six (6) groups consisted of n = 10. The stage of 45 min hypoxia was common for all six groups. Afterward, reperfusion of 60 min was followed in Group A; reperfusion of 120 min in Group B; immediate Epo intravenous (IV) administration and reperfusion of 60 min in Group C; immediate Epo IV administration and reperfusion of 120 min in Group D; immediate U-74389G IV administration and reperfusion of 60 min in Group E; and immediate U-74389G IV administration and reperfusion of 120 min in Group F. The dose height assessment for both drugs is described at preliminary studies as 10 mg/kg body mass.

Ischemia was caused by laparotomic clamping the inferior aorta over renal arteries with forceps for 45 min. The clamp removal was restoring the inferior aorta patency and reperfusion. After exclusion of the blood flow, the protocol of IR was applied, as described above for each experimental group. The drugs were administered at the time of reperfusion, through inferior vena cava catheter. The ASTl was determined at 60th min of reperfusion (for A, C, and E) and 120th min of reperfusion (for Groups B, D and F). Along, a weak relation was rised between ASTl values with animals' mass (P = 0.2154), so it was no use of calculating the predicted ASTl values for the animals' mass.

Statistical analysis
Table 1 presents the (%) hypertransferasemic influence of Epo regarding reoxygenation time. Furthermore, Table 2 presents the (%) hypertransferasemic influence of U-74389G regarding reperfusion time. Chi-square tests were applied using the ratios which produced the (%) results per endpoint. The outcomes of Chi-square tests are depicted in Table 3. The statistical analysis was performed by Stata 6.0 software (Stata 6.0, StataCorp LP, Texas, USA).

Table 1    Table 2    Table 3

RESULTS

---

The successive application of Chi-square tests revealed that U-74389G augmented the ASTl by 1.149264-fold (1.006956-1.311683) more than Epo at 1 h (P = 0.0391), by 0.9347365-fold (0.9334662-0.9360085) less than Epo at 1.5 h (P = 0.0000), by 0.6695775-fold (0.5800545-0.7729184) less than Epo at 2 h (P = 0.0000), less by 0.7631082 (0.7620376-0.7641804) (P = 0.0000) without drugs, and by 0.8224656-fold (0.8211631-0.8237701) less than Epo whether all variables have been considered (P = 0.0000).

DISCUSSION

---

The unique available study investigating the hypertransferasemic effect of U-74389G on ASTl was the preliminary one[1]. Apart from the most famous activities this drug has as the neuroprotection and membrane-stabilization properties, it also accumulates in the cell membrane, protects the vascular endothelium from peroxidative damage, but hardly penetrates the blood-brain barrier. It elicits a beneficial effect in ototoxicity and Duchenne muscular dystrophy. It increases γgt, superoxide dismutase, and glutathione levels in oxygen-exposed cells. It treats septic states and acts as immunosuppressant in flap survival. It prevents the learning impairments; it delays the early synaptic transmission decay during hypoxia improving energetic state of neurons. It shows antiproliferative properties on brain cancer cells and is considered as a new promising anti-inflammatory drug for the treatment of reperfusion syndrome in IR injuries.

The same authors confirmed[2] the short-term hypertransferasemic effect of Epo preparations in non-iron-deficient individuals. Li et al. demonstrated[3] that EPO may play a protective role against LPS-induced multiple organ failure by reducing the inflammatory response and tissue degeneration, possibly through the phosphatidylinositol 3-kinase/AKT and NF-kB signaling pathways restoring the LPS-induced ASTl in rats. Fu et al. examined[4] the protective effect of rHuEPO which is mediated through the activation of the phosphatidylinositol-3 kinase/AKT/endothelial nitric oxide synthase (eNOS) signaling pathway, at least in part, by increasing p-AKT and p-eNOS, and leads to the maintenance of an elevated level of NO in I/R injury of the liver. Yildar et al. investigated[5] the protective effect of 2-aminoethyl diphenylborinate which significantly reduced the sAST in the rat kidney I/R injury group. Bayramoglu et al. examined[6] the use of lycopene, while improvements of the AST values were partial and dose-dependent (P < 0.05) in IR injury of rat liver. Liu et al. showed[7] that quercetin significantly reduced apoptosis rate, improved cardiac function, and decreased the levels of AST, by inhibiting apoptosis in vivo and PI3K/AKT pathway involved in the anti-apoptotic effect in SD rats in myocardial IR injuries in vivo. Bayramoglu et al. noticed[8] that gallic acid significantly decreased the AST activities in tissue homogenates than no treatment group (P < 0.05) in oxidative stress generated by hepatic I/R-induced control rats. Matsuno et al. found[9] the ASTl in the control versus hydrogen group in 30, 60, and 120 min after reperfusion being more by 8.9%, 9.59%, and 3.54%, respectively, in a porcine liver reperfusion injury. Saidi et al. compared[10] the control group with animals treated with tilapia fish oil which experienced a significant decrease (P < 0.05) in ASTl in reperfusion periods in male Wistar rats subjected to warm liver IRI. Li et al. significantly ameliorated[11] the I/R injury of the liver after Mino treatment, as shown by decreased Suzuki scores and liver function AST in male Sprague-Dawley rats liver. Ozkan et al. concluded[12] that hypothermic reperfusion and O₃ preconditioning might be beneficial in skeletal muscle IR injury since the ASTl were decreased than those in the IR group in the rats. Hu et al. found[13] the AST activities declined (P < 0.05) in tanshinone IIA (TSA) L-TSA, M-TSA, and H-TSA rat myocardial ischemia groups. Cahova et al. indicated[14] that metformin treatment prevented an acute stress-induced necroinflammatory reaction, reduced AST serum activity, diminished lipoperoxidation, and reduced mitochondrial performance but concomitantly protected the liver from I/R-induced injury in Wistar rats. Yildiz et al. showed[15] a decrease in ASTl in the micronized purified flavonoid fraction-treated rats than hepatic I/R group rats (P < 0.001 for all). Zhang et al. indicated[16] that hydrogen inhalation at 2% concentration for 1 h before liver syngeneic orthotopic transplantation protected from ischemia/reperfusion injury by activation of the NF-kB signaling pathway, reducing the sAST activities in rats. Lee et al. found[17] hepatocytes AlbCre+/constitutively active nuclear factor (erythroid-derived 2)-like 2 (caNrf2)+ having significantly reduced serum transaminases than wild-type littermate controls in warm hepatic murine IRI. Tang et al. regarded[18] Dioscorea nipponica Makino, D. panthaica Prain et Burkill (DP), and D. zingiberensis C.H. Wright (DZ) as having the same traditional therapeutic actions, such as TS groups exhibiting significantly reduced activities of AST than the model group (ISO injection only). Yucel et al. noticed that infliximab significantly reduced[19] the ASTl in IR liver. Deng et al. significantly reduced the levels of AST[20] in the MT group at each time point than that of the liver function IR group (P < 0.05) in male Sprague-Dawley rats. Lee et al. acknowledged that[21] treatment with eupatilin significantly decreased sAST as well as liver histologic changes in acute IR-induced hepatic damage. Ozsoy et al. found remarkably higher sASTl[22] in IR group than the sham group and the laboratory tests returned to normal levels in IR+melatonin (MEL) group after MEL treatment of the hepatic tissue. Bektas et al. found sAST[23] significantly different in tadalafil and pentoxifylline groups than liver IR group. Younis et al. investigated[24] the Silymarin preconditioning which decreased AST and improved hepatic architecture in HIR injury of insulin-resistant rats. Abdelsameea et al. pretreated with liraglutide which decreased[25] AST activities with attenuation of necrosis and inflammation while enhanced Bcl-2 expression in liver IR male rats. Liu et al. shown[26] that the formula of Guanxin Dhutong capsule which has four main active ingredients, protocatechuic acid, cryptotanshinone, borneol, and eugenol, diminished the infarct size and reduced ASTl in vivo in a rat model of the coronary artery. Hao et al. noticed that propofol significantly reduced[27] the activities of sAST in QSG-7701 cells in vitro of hepatic I/R injury in rats. Wang et al. revealed[28] that 2'-O-galloylhyperin effectively ameliorated CCl4-induced hepatic damage by reducing AST activities in an animal model. Saidi et al. invented that Pistacia lentiscus oil decreased[29] both the visible severe intestinal damage and the sASTl in intestinal IR. Cai et al. pretreated with salidroside (20 mg/kg/day for 7 days, intraperitoneally) which significantly decreased[30] sAST levels after 6 h and 24 h of reperfusion and protected the segmental (70%) warm hepatic liver against I/R-induced injury. Wang et al. found[31] that flavonol glycosides which are in high concentrations in Inula racemosa altered cellular morphology, and cytokines and AST were returned to near normal level in hepatic I/R injury. Ardasheva et al. elevated the intra-abdominal pressure (IAP) above 20 mmHg which led[32] to progression of abdominal compartment syndrome that is associated with organ dysfunction or failure and elevated activities of AST found in some of the experimental groups than control ones of animals not subjected to increased IAP and time frame tested for liver. Zazueta et al. found[33] that citicoline CDP-choline reduced ASTl in blood samples from reperfused rats in I/R rat livers. Miyauchi et al. evaluated the levels of sAST as well as the scores of liver necrosis[34] significantly lower in the antioxidative nutrient-rich enteral diet (Ao diet) group than the control diet group in liver IR mice. Zhang et al. showed that[35] the role of hydrogen-rich saline treatment significantly decreased serum levels of AST activity and TUNEL-positive cells in miniature pig model of laparoscopic HIRI on hepatectomy. Chen et al. observed[36] significantly decreased (all P < 0.05) the levels of AST in the rosiglitazone group than the HIRI group in a rat model of hepatic ischemia-reperfusion injury. Gholampour et al. treated[37] with remote ischemic perconditioning and quercetin which reduced plasma AST activity in the liver after renal I/R. Brandao et al. observed[38] separately the protective effects of allopurinol and the benefits of ischemic post-conditioning by measuring ASTl with significantly reduced I/R systemic injuries in rats.

According to above, Table 3 shows that U-74389G has 0.8224656-fold (0.8211631-0.8237701) less hypertransferasemic effect than Epo (P = 0.0000) whether all variables have been considered (P = 0.0000), a trend attenuated along time, in Epo non-deficient rats. A meta-analysis of these ratios from the same experiment, for 20 other seric variables, provides comparable results [Table 4][39,40].

Table 4

CONCLUSION

---

The antioxidant agent U-74389G was proved having 0.8224656-fold (0.8211631-0.8237701) less hypertransferasemic effect than Epo whether all variables have been considered (P = 0.0000); a trend attenuated along the short-term time frame of the experiment in rats. A biochemical investigation remains about how U-74389G mediates in these actions.

ACKNOWLEDGMENT

---

This study was funded by Scholarship by the Experimental Research Center ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece. The research facilities for this project were provided by the aforementioned institution.

REFERENCES

---

1. Tsompos C, Panoulis C, Toutouzas K, Triantafyllou A, Zografos G, Papalois A. The effect of the antioxidant drug "U-74389g" on aspartate aminotransferase levels during ischemia reperfusion injury in rats. J Biotechnol Sci Res 2017;4:198-203.
2. Tsompos C, Panoulis C, Toutouzas K, Triantafyllou A, Zografos G, Papalois A. The effect of erythropoietin on aspartate aminotransferase levels during ischemia reperfusion injury in rats. Electron J Biol 2016;12:161-7.
3. Li XJ, Zhang GX, Sun N, Sun Y, Yang LZ, Du YJ, et al. Protective effects of erythropoietin on endotoxin-related organ injury in rats. J Huazhong Univ Sci Technolog Med Sci 2013;33:680-6.
4. Fu W, Liao X, Ruan J, Li X, Chen L, Wang B, et al. Recombinant human erythropoietin preconditioning attenuates liver ischemia reperfusion injury through the phosphatidylinositol-3 kinase/ AKT/endothelial nitric oxide synthase pathway. J Surg Res 2013;183:876-84.
5. Yildar M, Aksit H, Korkut O, Ozyigit MO, Sunay B, Seyrek K, et al. Protective effect of 2-aminoethyl diphenylborinate on acute ischemia-reperfusion injury in the rat kidney. J Surg Res 2014;187:683-9.
6. Bayramoglu G, Bayramoglu A, Altuner Y, Uyanoglu M, Colak S. The effects of lycopene on hepatic ischemia/ reperfusion injury in rats. Cytotechnology 2015;67:487-91.
7. Liu H, Guo X, Chu Y, Lu S. Heart protective effects and mechanism of quercetin preconditioning on anti-myocardial ischemia reperfusion (IR) injuries in rats. Gene 2014;545:149-55.
8. Bayramoglu G, Kurt H, Bayramoglu A, Gunes HV, Degirmenci I, Colak S, et al. Preventive role of gallic acid on hepatic ischemia and reperfusion injury in rats. Cytotechnology 2015;67:845-9.
9. Matsuno N, Watanabe R, Kimura M, Iwata S, Fujiyama M, Kono S, et al. Beneficial effects of hydrogen gas on porcine liver reperfusion injury with use of total vascular exclusion and active venous bypass. Transplant Proc 2014;46:1104-6.
10. Saidi SA, Abdelkafi S, Jbahi S, van Pelt J, El-Feki A. Temporal changes in hepatic antioxidant enzyme activities after ischemia and reperfusion in a rat liver ischemia model: Effect of dietary fish oil. Hum Exp Toxicol 2015;34:249-59.
11. Li Y, Li T, Qi H, Yuan F. Minocycline protects against hepatic ischemia/reperfusion injury in a rat model. Biomed Rep 2015;3:19-24.
12. Ozkan H, Ekinci S, Uysal B, Akyildiz F, Turkkan S, Ersen O, et al. Evaluation and comparison of the effect of hypothermia and ozone on ischemia-reperfusion injury of skeletal muscle in rats. J Surg Res 2015;196:313-9.
13. Hu H, Zhai C, Qian G, Gu A, Liu J, Ying F, et al. Protective effects of tanshinone IIA on myocardial ischemia reperfusion injury by reducing oxidative stress, HMGB1 expression, and inflammatory reaction. Pharm Biol 2015;53:1752-8.
14. Cahova M, Palenickova E, Dankova H, Sticova E, Burian M, Drahota Z, et al. Metformin prevents ischemia reperfusion-induced oxidative stress in the fatty liver by attenuation of reactive oxygen species formation. Am J Physiol Gastrointest Liver Physiol 2015;309:G100-11.
15. Yildiz F, Coban S, Terzi A, Aksoy N, Bitiren M. Protective effect of micronized purified flavonoid fraction on ischemia/ reperfusion injury of rat liver. Transplant Proc 2015;47:1507-10.
16. Zhang CB, Tang YC, Xu XJ, Guo SX, Wang HZ. Hydrogen gas inhalation protects against liver ischemia/reperfusion injury by activating the NF-kB signaling pathway. Exp Ther Med 2015;9:2114-20.
17. Lee LY, Harberg C, Matkowskyj KA, Cook S, Roenneburg D, Werner S, et al. Overactivation of the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element pathway in hepatocytes decreases hepatic ischemia/reperfusion injury in mice. Liver Transpl 2016;22:91-102.
18. Tang YN, He XC, Ye M, Huang H, Chen HL, Peng WL, et al. Cardioprotective effect of total saponins from three medicinal species of Dioscorea against isoprenaline-induced myocardial ischemia. J Ethnopharmacol 2015;175:451-5.
19. Yucel AF, Pergel A, Aydin I, Alacam H, Karabicak I, Kesicioglu T, et al. Effect of infliximab on acute hepatic ischemia/reperfusion injury in rats. Int J Clin Exp Med 2015;8:21287-94.
20. Deng WS, Xu Q, Liu YE, Jiang CH, Zhou H, Gu L, et al. Effects of melatonin on liver function and lipid peroxidation in a rat model of hepatic ischemia/reperfusion injury. Exp Ther Med 2016;11:1955-60.
21. Lee HM, Jang HJ, Kim SS, Kim HJ, Lee SY, Oh MY, et al. Protective effect of eupatilin pretreatment against hepatic ischemia-reperfusion injury in mice. Transplant Proc 2016;48:1226-33.
22. Ozsoy M, Gonul Y, Ozkececi ZT, Bali A, Celep RB, Kocak A, et al. The protective effect of melatonin on remote organ liver ischemia and reperfusion injury following aortic clamping. Ann Ital Chir 2016;87:271-9.
23. Bektas S, Karakaya K, Can M, Bahadir B, Guven B, Erdogan N, et al. The effects of tadalafil and pentoxifylline on apoptosis and nitric oxide synthase in liver ischemia/reperfusion injury. Kaohsiung J Med Sci 2016;32:339-47.
24. Younis NN, Shaheen MA, Mahmoud MF. Silymarin preconditioning protected insulin resistant rats from liver ischemia-reperfusion injury: Role of endogenous H2S. J Surg Res 2016;204:398-409.
25. Abdelsameea AA, Abbas NA, Abdel Raouf SM. Liraglutide attenuates partial warm ischemia-reperfusion injury in rat livers. Naunyn Schmiedebergs Arch Pharmacol 2017;390:311-9.
26. Liu F, Huang ZZ, Sun YH, Li T, Yang DH, Xu G, et al. Four main active ingredients derived from a traditional Chinese medicine guanxin shutong capsule cause cardioprotection during myocardial ischemia injury calcium overload suppression. Phytother Res 2017;31:507-15.
27. Hao W, Zhao ZH, Meng QT, Tie ME, Lei SQ, Xia ZY, et al. Propofol protects against hepatic ischemia/reperfusion injury via miR-133a-5p regulating the expression of MAPK6. Cell Biol Int 2017;41:495-504.
28. Wang P, Gao YM, Sun X, Guo N, Li J, Wang W, et al. Hepatoprotective effect of 2'-O-galloylhyperin against oxidative stress-induced liver damage through induction of nrf2/ARE-mediated antioxidant pathway. Food Chem Toxicol 2017;102:129-42.
29. Saidi SA, Ncir M, Chaaben R, Jamoussi K, van Pelt J, Elfeki A, et al. Liver injury following small intestinal ischemia reperfusion in rats is attenuated by Pistacia lentiscus oil: Antioxidant and anti-inflammatory effects. Arch Physiol Biochem 2017;123:199-205.
30. Cai L, Li Y, Zhang Q, Sun H, Yan X, Hua T, et al. Salidroside protects rat liver against ischemia/reperfusion injury by regulating the GSK-3β/Nrf2-dependent antioxidant response and mitochondrial permeability transition. Eur J Pharmacol 2017;806:32-42.
31. Wang Z, Geng L, Chen Z, Lin B, Zhang M, Zheng S, et al. In vivo therapeutic potential of Inula racemosa in hepatic ischemia-reperfusion injury following orthotopic liver transplantation in male albino rats. AMB Express 2017;7:211.
32. Ardasheva RG, Argirova MD, Turiiski VI, Krustev AD. Biochemical changes in experimental rat model of abdominal compartment syndrome. Folia Med (Plovdiv) 2017;59:430-6.
33. Zazueta C, Buelna-Chontal M, Macias-Lopez A, Roman-Anguiano NG, Gonzalez-Pacheco H, Pavon N, et al. Cytidine-5'-diphosphocholine protects the liver from ischemia/ reperfusion injury preserving mitochondrial function and reducing oxidative stress. Liver Transpl 2018;24:1070-83.
34. Miyauchi T, Uchida Y, Kadono K, Hirao H, Kawasoe J, Watanabe T, et al. Preventive effect of antioxidative nutrient-rich enteral diet against liver ischemia and reperfusion injury. JPEN J Parenter Enteral Nutr 2018;31:181-7.
35. Zhang Q, Ge Y, Li H, Bai G, Jiao Z, Kong X, et al. Effect of hydrogen-rich saline on apoptosis induced by hepatic ischemia reperfusion upon laparoscopic hepatectomy in miniature pigs. Res Vet Sci 2018;119:285-91.
36. Chen J, Liu H, Zhang X. Protective effects of rosiglitazone on hepatic ischemia reperfusion injury in rats. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2018;43:732-7.
37. Gholampour F, Sadidi Z. Hepatorenal protection during renal ischemia by quercetin and remote ischemic perconditioning. J Surg Res 2018;231:224-33.
38. Brandao RI, Gomes RZ, Lopes L, Linhares FS, Vellosa JC, Paludo KS, et al. Remote post-conditioning and allopurinol reduce ischemia-reperfusion injury in an infra-renal ischemia model. Ther Adv Cardiovasc Dis 2018;12:341-9.
39. Tsompos C, Panoulis C, Toutouzas K, Triantafyllou A, Zografos CG, Tsarea K, et al. Comparison of the hyperphosphatasemic effects of erythropoietin and U-74389G on alkaline phosphatase levels. Int J Res Stud Sci Eng Technol 2018;5:7-13.
40. Tsompos C, Panoulis C, Toutouzas K, Triantafyllou A, Zografos CG, Tsarea K, et al. The diverging effects of erythropoietin and U-74389gon γ-glutamyl transferase levels. Arch Nephrol 2018;1:31-5.