Comparison of the Hyperkinasemic Effects of Erythropoietin and U-74389G on Creatine Phosphokinase Levels


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Citation: Comparison of the Hyperkinasemic Effects of Erythropoietin and U-74389G on Creatine Phosphokinase Levels. American Research Journal of Biomedical Engineering. vol 2, no. 1: 1-7

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Abstract:

Aim: This study calculated the effects on creatine phosphokinase (CPK) levels, after treatment with either of 2 drugs: the erythropoietin (Epo) and the antioxidant lazaroid (L) drug U-74389G. The calculation was based on the results of 2 preliminary studies, each one of which estimated the certain influence, after the respective drug usage in an induced ischemia reperfusion (IR) animal experiment.

Materials and Methods: The 2 main experimental endpoints at which the serum CPK levels (CPKl) were evaluated was the 60th reperfusion min (for the groups A, C and E) and the 120th reperfusion min (for the groups B, D and F). Specially, the groups A and B were processed without drugs, groups C and D after Epo administration; whereas groups E and F after the L administration.

Results: The first preliminary study of Epo presented a non significant hyperkinase mice ffect by 2.08%+2.77% (p-value=0.4430). The second preliminary study of U-74389G presented a significant hyperkinasemic effect by 8.52%+4.35% (p-value=0.0005). These 2 studies were co-evaluated since they came from the same experimental setting. The outcome of the co-evaluation was that L is 4.09626-fold [4.092989 - 4.099534] more hyperkinasemic than Epo (p-value=0.0000).

Conclusions: The anti-oxidant capacities of U-74389G ascribe 4.09626-fold more hyperkinasemic effects than Epo (p-value=0.0000).

Keywords: ischemia; erythropoietin; U-74389G; creatine phosphokinase levels; reperfusion


Description:

INTRODUCTION

The lazaroid U-74389G (L) may be not famous for its hyperkinasemic1 capacity (p-value=0.0005). U-74389G as a novel antioxidant factor, implicates exactly only 260 published studies. The ischemia reperfusion (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 acid-induced one. Animal kidney, liver, brain microvascular endothelial cells monolayers and heart models were protected by U-74389G after IR injury. U-74389G also attenuates the leukocytes; down-regulates the proinflammatory gene; treats the endotoxin shock; produces cytokine; enhances the mononuclear immunity; protects the endothelium and presents antishock property.

Erythropoietin (Epo) even if is not famous for itshyperkinasemic action (p-value=0.4430), it can be used as a reference drug for comparison with U-74389G. Although Epo is met in over 30,637 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 oncreatine phosphokinaselevels (CPK) 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 serumCPK levels (CPKl) reductions.

MATERIALS AND METHODS

Animal Preparation

The Vet licenses under 3693/12-11- 2010 & 14/10-1-2012 numbers, the granting company and the experiment location are mentioned in preliminary references1,2. The human animal care of Albino female Wistar rats, the 7 days pre-experimental ad libitum diet, the non-stop intra-experimental anesthesiologic techniques, the acidometry, the electrocardiogram, the 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 in N=10. The stage of 45 min hypoxia was common for all 6 groups. Afterwards, 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 are 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 CPKlwere determined at 60th min of reperfusion (for A, C and E groups) and at 120th min of reperfusion (for B, D and F groups). Along, due to a strong relation was rised between CPKl values with animals’ mass (p-value=0.0105), the predicted CPKl values were used.

Statistical Analysis

Table 1 presents the (%) hyperkinasemic influence of Epo regarding reoxygenation time. Also, Table 2 presents the (%) hyperkinasemic 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 at Table 3. The statistical analysis was performed by Stata 6.0 software [Stata 6.0, StataCorp LP, Texas, USA].

RESULTS

The successive application of chi-square tests revealed that U-74389G enhanced the CPKl by 144.0769-fold [143.9114 - 144.2425] more than Epo at 1h (p-value=0.0000), by 3.987264-fold [3.979742 - 3.9948] more than Epo at 1.5h (p-value=0.0000), by 2.567192-fold [2.563487 - 2.570902] more than Epo at 2h (p-value=0.0000), less by 0.7974539-fold [0.7954859 - 0.7994269] (p-value=0.0000) without drugs and by 4.09626-fold [4.092989 - 4.099534] more than Epo whether all variables have been considered (p-value=0.0000).

DISCUSSION

The unique available study investigating the hyperkinasemic effect of U-74389G on CPKl was the preliminary one1. Although the most famous activities of neuroprotection and membrane-stabilization properties, it accumulates in the cell membrane, protecting 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 (SOD) and glutathione (GSH) 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 confirmed2 the short-termhyperkinasemiceffect of Epo preparations in non iron deficient individuals. Quan W et al showed3 that Magnesium lithospermate B (MLB) significantly increased phosphorylation of Akt and that this phosphorylation can be partially inhibited by phosphoinositide 3-kinase/Akt inhibitor. The results also showed that MLB prevents I/R-induced myocardial damage by reducing necrosis and apoptosis in H9c2 cardiomyocytes, improving myocardial function in rat hearts. Yoshino T et al concluded4 a temporal induction of preconditioning actions of the aldosterone cascade, at a physiological dose, having favorable effects on cardiac functional recovery in left ventricular contractility and left ventricular end-diastolic pressure associated4 with a reduced activity of creatine phosphokinase released into the perfusate after injury following ischemia-reperfusion in a MR-independent manner male in Wistar rat Langendorff hearts. Takhtfooladi H et al alleviated5 the metabolic injuries in the skeletal muscle ischemia and reperfusion in this experimental model after tramadol treatment in a rat hind limb ischemia-reperfusion model. Dianat M et al concluded6 at a more significant decrease in the CPK levels in groups that had received combined treatment in comparison with vanillic acid (antioxidant) or losartan (selective angiotensin II (ANG-II) type 1 receptor (AT1R) blocker alone and hence, protects myocardium against I/R-induced oxidative stress injuries in isolated rat hearts. Liao Z et al strikingly found7 that resveratrol downregulates voltage-dependent anion channel 1 (VDAC1), leading to prevention of mitochondrial permeability transition pore opening and cardiomyocyte apoptosis; decreased the creatine phosphokinase activities; and reduced the infarction size. The data also revealed that long-term oral intake of resveratrol sets nutritional preconditioning to cope with myocardial I/R injury. Zhang N et al showed8 that preoperative trimetazidine therapy appears to have a positive effect on myocardial preservation along with significantly lower postoperative levels of CK, TnT and TnI, also in both the ≤12 and >12 h subgroup analyses in control coronary artery bypass grafting (CABG) patients. Lemarié J et al hypothesized9 that the triggering receptor expressed on myeloid cells (TREM)-1 acts as an amplifier of the immune response triggered by toll-like receptor engagement. TREM-1 inhibition by inhibitory peptide LR12 significantly improved these dysfunctions (P<0.03) and restricted infarct size, as assessed by lower creatine phosphokinase and troponin I concentrations (P><0.005) in a clinically relevant porcine model of acute myocardial infarction. Kashiwagi Y et al provided10 new insight into the significant role of sodium-glucose cotransporter 1 (SGLTs) in optimizing cardiac energy metabolism, at least during the acute phase of IRI, whether it is expressed in human hearts and significantly contributes to cardiac energy metabolism during ischemia-reperfusion injury (IRI) via enhanced glucose utilization. Phlorizin administration during IRI significantly impaired the recovery in left ventricular contractions and rate pressure product, associated with an increased infarct size, as demonstrated by triphenyltetrazolium chloride staining and creatine phosphokinase activity released into the perfusate in mice. Li CM et al demonstrated11 that the cardioprotective effect of ischemic post-conditioning was involved in the inhibition of PTEN, activation of the PI3K/Akt signal pathway and reduction of the cardiomyocyte apoptosis confirmed by serum creatine phosphokinase activity.Luo SY et al indicated12 that SB-710411 - a rat selective urotensin-II (U-II) receptor antagonist, which can block U-II-induced contraction of the aorta - inhibits U-IIinduced myocardial fibrosis and decreases myocardial UTR expression in myocardiac I/R injury of rats. Qiu LY et al revealed13 that treatment with SQS promoted protein kinase C ε (PKCε) which is able to mediate Cl‑ homeostasis - phosphorylation and inhibited sI/R-induced elevation of [Cl-]i, paralleled by the attenuation of mitochondrial membrane potential loss and ROS generation; eliciting cardioprotectionincreasing the viability and efficiently attenuating the creatine phosphokinase release in cultured cardiomyocytes. Takhtfooladi HA et al found significantly lower serum CPK levels14 (P><0.05) after low-level laser therapy (LLLT) which has a beneficial effect on the IR gastrocnemius muscle injury treatment in streptozotocin-induced diabetic male Wistar rats. Claroni C et al found15 the positive preconditioning impact of sevoflurane on IR injury expressed in the early postoperative hours, but not in the long-term ones since the creatine phosphokinase value was significantly lower in the BAL group only at the end of operation and not at other endpoints in patients undergoing free flap operation. Fujisaki N et al protected16 cardiac grafts from ischemia reperfusion-induced injury after pretreatment with CO gas before organ procurement effectively. Recipients of grafts from COexposed donors had lower levels of serum troponin I and creatine phosphokinase; less upregulation of mRNA for interleukin-6, intercellular adhesion molecule-1, and tumor necrosis factor-α; and fewer infiltrating cells. Donor pretreatment with CO altered the expression of 49 genes expressly represented on the array in a rat heterotopic cardiac transplant model. Kundra TS et al prevented17 skeletal muscle ischemia-reperfusion injury in patients undergoing aortobifemoral bypass operation after dexmedetomidine administration and CPK values assessments. Zeng G et al demonstrated18 that SIRT4, a mitochondrial-localized sirtuin is downregulated in cardiomyocytes both in vitro and in vivo models after MI-R. Functionally, SIRT4 overexpression decreases myocardial infarct size and serum creatine phosphokinase (CPK) level, and vice versa, SIRT4 depletion by siRNA increases myocardial infarct size and serum CPK level. Yin TC et al concluded19 that extracorporeal shock wave (ECSW) - adipose-derived mesenchymal stem cells (ADMSC) therapy is superior to either one applied individually since the microscopic findings of endothelial-cell biomarkers and number of arterioles expressedan opposite pattern of CPK, whereas the histopathology showed that muscle-damaged/fibrosis/ collagen-deposition areas exhibited an identical pattern of CPK among the five groups (all p>< 0.0001) for protecting against IR-induced thigh injury in rats.

According to above, table 3 shows that U-74389G has 4.09626-fold [4.092989 - 4.099534] morehyperkinasemic effect than Epo (p-value=0.0000) whether all variables have been considered (p-value=0.0000); a trend attenuated along time, in Epo non-deficient rats. A meta-analysis of these ratios from the same experiment, for 21 other seric variables, provides comparable results (table 4)20.