Accepted Manuscript Effect of Otostegia Persica extraction on renal injury induced by hindlimb ischemiareperfusion: a rat model Mohammad Ashrafzadeh Takhtfooladi , Hamed Ashrafzadeh Takhtfooladi , Fariborz Moayer , Poorya Karimi , Hesam Abbasian Asl PII:
S1743-9191(14)01010-3
DOI:
10.1016/j.ijsu.2014.11.047
Reference:
IJSU 1673
To appear in:
International Journal of Surgery
Received Date: 1 March 2014 Revised Date:
24 November 2014
Accepted Date: 29 November 2014
Please cite this article as: Takhtfooladi MA, Takhtfooladi HA, Moayer F, Karimi P, Asl HA, Effect of Otostegia Persica extraction on renal injury induced by hindlimb ischemia-reperfusion: a rat model, International Journal of Surgery (2015), doi: 10.1016/j.ijsu.2014.11.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Effect of Otostegia Persica extraction on renal injury induced by hindlimb ischemia-
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reperfusion: a rat model
Mohammad Ashrafzadeh Takhtfooladi1, Hamed Ashrafzadeh Takhtfooladi2, Fariborz Moayer3, Poorya Karimi4,
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Hesam Abbasian Asl4
1
Young Researchers and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2
Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
3
Department of Pathobiology, College of Veterinary Medicine, Karaj Branch, Islamic Azad University, Alborz, Iran.
4
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Department of Clinical Sciences, College of Veterinary Medicine, Karaj Branch, Islamic Azad University, Alborz, Iran.
Correspondence to: Mohammad Ashrafzadeh Takhtfooladi. Young Researchers and Elites Club, Science and
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Research Branch, Islamic Azad University, Tehran, Iran. Tel. 00989121590428. Fax: 00983117860211. E-mail:
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[email protected]
ACCEPTED MANUSCRIPT ABSTRACT Introduction: It is known that ischemia-reperfusion causes remote organ injury as well as local injury. In traditional systems of medicine, many plants have been documented to be useful for the treatment of various disorders including oxidative esters. This study was designed to investigate whether Otostegia
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persica extraction pretreatment has a protective effect against renal injury induced by hindlimb ischemiareperfusion. Methods: Forty male Wistar rats were allocated into five groups as follows: Control, Sham, Otostegia persica, ischemia-reperfusion and ischemia-reperfusion + Otostegia persica groups. Rats in Otostegia persica and ischemia-reperfusion + Otostegia persica groups received Otostegia persica
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extraction (300 mg/kg) orally 2 days prior to operation. Hindlimb ischemia was induced by clamping the femoral artery for 2 hours. After 24 hours of reperfusion, blood and urine samples were obtained for
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kidney function tests and the kidneys were removed for histological analysis and oxidative stress measurement. Results: The decrease in glomerular filtration rate induced by reperfusion was significantly improved by Otostegia persica extraction administration (P < 0.05), which resulted in the decrease in serum urea and creatinine concentrations. Urinary creatinine significantly decreased in ischemiareperfusion group compared to the other groups (P < 0.05). Urinary excretion rate, water intake and the
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ratio of kidney/body weight significantly increased in animals with reperfusion injury as compared with other groups (P < 0.05). On histological examination, rats pretreated with Otostegia persica extraction had nearly normal morphology. Skeletal muscle ischemia-reperfusion produced a significant increase in
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renal tissue malondialdehyde level, while pretreatment with Otostegia persica extraction was associated with a significantly lower malondialdehyde level (P < 0.05). Renal tissue catalase and superoxide dismutase activity and glutathione level were significantly (P < 0.05) decreased by hindlimb ischemia-
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reperfusion. The increases in these parameters were decreased by pretreatment with Otostegia persica extraction. Conclusions: The results of this study showed that Otostegia persica extraction pretreatment significantly protected the renal injury from skeletal muscle ischemia-reperfusion.
Keywords: skeletal muscle; ischemia-reperfusion; remote organ damage; kidney injury; Otostegia persica.
ACCEPTED MANUSCRIPT 1. Introduction Skeletal muscle ischemia-reperfusion injury is common in several clinical practice including trauma, disease and orthopedic surgery.1 Reperfusion injury of the extremities is characterized by metabolic acidosis, increased serum creatine kinase, myoglobinury, hyperkalemia by the loss of intracellular
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potassium and free radicals release.2 Numerous free radicals are brought along when oxygenated blood flow re-enters ischemic tissues during reperfusion, leading to additional tissue injury.3
The development of remote organ dysfunction was observed only following reperfusion, which implies that cellular mediators produced locally in the limb were responsible for mediating remote organ
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injury.4,5 Histological and biochemical evidences of lung, liver and kidney dysfunction indicates that multiple organ dysfunctions following hindlimb ischemia-reperfusion occur as a central systemic event
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rather than sequential failure of individual organs.5-7
Multiple pharmacological agents such as iloprost,8 vitamin C,8 pentoxifylline,9 L-alanyl-glutamin,10 N-acetylcysteine11 and erythropoietin12 are proposed to be useful against renal remote injury after hindlimb ischemia-reperfusion.
Otostegia persica (Burm.) Boiss., that locally known as “Golder” is an endemic medicinal plant
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growing in southern and also southeastern of Iran.13 The extract of the Otostegia persica is used in Iranian traditional medicine as anti-histaminic, anti-spasmodic, anti-arthritis, anti-pyretic and analgesic in toothache.14,15 There are evidences that Otostegia persica extraction also is an effective treatment for saving the burn site.16 The aerial part of Otostegia persica is reported to have high antioxidant activity
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which is related to the flavonoids.17 Additional biological screening of the methanolic extract has revealed strong antioxidant as well as antibacterial activities against various strains of Gram-negative and Gram-
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positive bacteria.17,18 According to experiments, it is determined that methanolic extract of the Otostegia persica has antioxidant properties.13 Despite many uses of Otostegia persica, no data can be found about the antioxidant role of the plant in reperfusion syndrome. This prompted us to carry out an investigation for effects of Otostegia persica on renal remote organ injury after hindlimb ischemia-reperfusion in rat model.
2. Material and methods
ACCEPTED MANUSCRIPT All experimental procedures were performed according to the guidelines for the ethical treatment of experimental animals and approved by Islamic Azad University School of Veterinary Science, Animal Care and Use Local Ethics Committee.
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2.1. Experimental protocol The sample size is calculated using the following formula:
where n is the required sample size. For Z α, Z is a constant (set by convention according to the accepted α
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error and whether it is a one-sided or two-sided effect) as shown below:
For Z1-β, Z is a constant set by convention according to power of the study as shown below:
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In the above-mentioned formula σ is the standard deviation (estimated) and ∆ the difference in effect of two interventions which is required (estimated effect size). Forty male Wistar rats weighing 300±35g were used in this study. All rats were maintained under
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constant room temperature of 25 ± 2°C, 12h/12h light/dark cycle with access to water and commercial food ad libitum and placed in individual cages. Animals were randomly allocated into five experimental
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groups of eight rats each:
Group I - Control group, with no operation and no treatment. Group II - Sham group with no ischemia-reperfusion. The animals were subjected to all operative procedures (isolation and exposure of the femoral artery for 2h) except arterial occlusion and reperfusion. Group III (Otostegia persica group) - which only received orally Otostegia persica extraction (300 mg/Kg)19 by using gavage technique, 2 days prior to operation. Group IV (ischemia-reperfusion group) - The animals were subjected to ischemia-reperfusion. Group V (ischemia-reperfusion+Otostegia persica group) - Otostegia persica extraction, 300 mg/kg, was orally administered 2 days prior to induction of ischemia-reperfusion.
ACCEPTED MANUSCRIPT Anesthesia was induced using intramuscular ketamine hydrochloride 10% (50 mg/kg) plus xylazine hydrochloride 2% (10 mg/kg). After the induction of anesthesia, 250 IU heparin was administered via the jugular vein to prevent clotting. Then, a skin incision (by using a sterile technique) was made on the medial surface of the left hindlimb. After the isolation of the femoral artery and vein from the
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surrounding tissues, the femoral artery was exposed. Hindlimb ischemia was induced by clamping the femoral artery. Rats were maintained in dorsal recumbency and kept anesthetized (additional doses were given as necessary) throughout the duration of the ischemic period. Body temperature was maintained with a heating pad and monitored using a rectal thermometer. After 2h ischemia, the vascular forceps was
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removed and surgical site was routinely closed. After surgery, fluid losses were replaced by administration of 5ml of warm (37˚C) isotonic saline i.p, and rats were kept in metabolic cages for 24h to
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collect urine and also to measure water consumption.
2.2 Plant material
The plant Otostegia persica was collected from Kerman province, Iran. The plant was identified by the Department of Botany of the Islamic Azad University. A voucher specimen has been deposited at the
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Herbarium of Department of Botany. Aerial parts of Otostegia persica were finely powdered in a mill. 500g of sample was selected and raised the volume to 1L by ethanol (96%). The solution percolated after 48h, then the solvent was removed under reduced pressure at low temperature and finally about 10g
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concentrated extract was prepared. Doses of the extract were prepared using normal saline.14,20
2.3. Biochemical analysis
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After reperfusion period, the blood samples were collected from jugular vein and were allowed to clot for 10 min at room temperature. Clots were centrifuged at 2500 rpm for 10 min to separate the serum and submitted for evaluation of serum creatinine and urea levels. Creatinine and urea concentrations of serum and urine creatinine were measured spectrophotometrically by using commercial kits by Automatic DADE AnalyzerTM.
2.4. Histological analysis
ACCEPTED MANUSCRIPT After euthanizing the animals by overdose of intraperitoneal pentobarbital injection (300 mg/kg), their kidneys were quickly removed. The kidneys from each rat were weighted for measurement of the ratio of kidney/body weight and the left kidneys were used for histological analysis under light microscopy. Renal tissues were placed in 10% formalin solution and processed according to standard procedures by
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embedding in paraffin then tissues were sectioned in 5µm pieces and stained with Hematoxylin-Eosin stain. Specimens were evaluated by one pathologist who was blinded to the experiment and data. All specimens were examined for 7 histological parameters including cellular vacuolation, apoptosis, interstitial edema, tubular dilatation, hyaline cast, polymorphonucleocytes in outer medulla, and
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medullary congestion on a semiquantitative scale of none (–), mild (+), moderate (++), and severe (+++).21 Tubular epithelial necrosis was graded as follow: normal histology (–), tubular cell swelling and
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nuclear condensation with up 1/3 of tubular profile exhibiting nuclear loss (+),tubular cell swelling and nuclear condensation with 1/3 to 2/3 of tubular profile exhibiting nuclear loss (++), greater than 2/3 of tubular profile showing nuclear loss (+++).21
2.5. Preparation of renal tissue homogenates
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Renal tissue homogenate and supernatant samples were prepared as described by Bhalodia et al.22 In brief, right kidneys perfused immediately with icecold hypertonic saline solution, and homogenized in chilled potassium chloride (1.17%). The homogenate was centrifuged at 10500g for 20 minutes at 4˚C to
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get the postmitochondrial supernatant, which was used to assay lipid peroxidation activity. 22
2.6. Measurement of lipid peroxidation
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For evaluating the status of oxidative stress, malondialdehyde (end product of lipid peroxidation) levels in renal tissue samples were measured. Malondialdehyde content in the renal tissue were measured by the method described by Ohkawa and colleagues.23 This is based on the measurement of thiobarbituric acid malondialdehyde absorbance. The reaction mixture consisted of 0.2 mL of 8.1% sodium lauryl sulphate, 1.5 mL of 20% acetic acid solution adjusted to a pH of 3.5 with sodium hydroxide, and 1.5 mL of 0.8% aqueous solution of thiobarbituric acid added to 0.2 mL of 10% (w/v) of postmitochondrial supernatant. The mixture was made up to 4.0 mL with distilled water and heated at 95°C for 60 minutes. After cooling with water, 1.0 mL of distilled water and 5.0 mL of the mixture of n-butanol: pyridine (15:1
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2.7. Measurement of antioxidant enzymes
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Catalase activity was measured by the method that described by Claiborne.25 The rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm. Superoxide dismutase activity was recognized according to the method of Kono.26 Nitro blue tetrazolium decline to blue formazan by superoxide anion was traced at 560 nm. Non-protein sulfhydryl, as a marker
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at 412 nm according to the method of Jollow et al.27
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for reduced glutathione, was measured by the reduction of Ellman’s reagent by -SH groups that was read
2.8. Statistics
Statistics were performed on SPSS software (version 18). Data were expressed as the mean±standard deviation and analyzed by one way ANOVA, with the significance of individual comparisons being assessed by Bonferroni test, and unpaired Student’s t-test was performed where appropriate. Differences
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were considered significant at P values less than 0.05.
3. Results
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During the study, two animals died; one rat from group II during anesthesia, and one rat from group IV in the following first hour after surgery (all were replaced immediately). There were no symptoms
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before death; dead rats underwent necropsy without significant findings.
3.1. Biochemical findings
Rats that underwent skeletal muscle ischemia-reperfusion exhibited significant increase in serum concentrations of urea and creatinine levels as compared with other groups (P < 0.05). Pretreatment with Otostegia persica extraction significantly decreased serum creatinine and urea levels compared with the ischemia-reperfusion group (P < 0.05) (Figures 1,2). In groups with reperfusion induction, urinary creatinine significantly decreased compared to other groups (P < 0.05) (Figure 3).
ACCEPTED MANUSCRIPT 3.2. Urinary excretion, water intake and kidney/body weight ratio Urinary excretion rate increased in rats with reperfusion injury as compared with the other rats (P < 0.05). It significantly decreased by pretreatment of Otostegia persica extraction in ischemia-reperfusion + Otostegia persica group (P < 0.05) (Figure 4). Water intake increased in animals with reperfusion injury
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as compared with rats in the ischemia-reperfusion + Otostegia persica group (P < 0.05), control and sham animals, and it significantly decreased by pretreatment of Otostegia persica extraction in the ischemiareperfusion + Otostegia persica group (P < 0.05) (Figure 5). Eventually, the ratio of kidney weight to body weight also increased in the ischemia-reperfusion group as compared with Otostegia persica,
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control and sham groups (P < 0.05). Pretreatment of Otostegia persica extraction decreased this ratio
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compared with no Otostegia persica extraction in the ischemia-reperfusion group (p < 0.05) (Figure 6).
3.3. Lipid peroxidation activity
Figure 7 illustrates that the malondialdehyde values of kidney tissues in the sham and Otostegia persica groups were not significantly different (P > 0.05); however, occlusion of the femoral artery for 2h and reperfusion for 24h increased the level of renal tissue malondialdehyde in the ischemia-reperfusion
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group in comparison with the sham group (P < 0.05). Otostegia persica extraction administration could significantly decrease the malondialdehyde values in the ischemia-reperfusion + Otostegia persica group compared with its corresponding values in the ischemia-reperfusion group (P < 0.05), yet it was still
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significantly different from the sham group (P < 0.05).
3.4. Antioxidant activity
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Rats in the ischemia-reperfusion group showed significantly decreased enzymatic activity of catalase (P < 0.05) (Figure 8), superoxide dismutase (P < 0.05) (Figure 9), and reduced glutathione (P < 0.05) (Figure 10) when compared with the other groups rats. These declining trends were significantly less in the group that received Otostegia persica extraction compared with those in the ischemia-reperfusion group.
3.5. Histological findings
ACCEPTED MANUSCRIPT The histological changes were summarized in the Table 1. The sham and Otostegia persica groups of rats did not show any renal morphological changes. By contrast, the kidneys of the rats with ischemiareperfusion showed significant pathologic changes of tubular cell swelling, tubular dilatation, interstitial edema, moderate to severe necrosis, cast formation in tubule and glomerul, glomerul fibrosis and
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hemorrhage (Figure 11). Apoptotic renal epithelial cells and nuclear changes consisting of karyolysis, picnosis, and karyorhexia were also notified in the ischemia-reperfusion group. Slight histological alterations were seen in the ischemia-reperfusion + Otostegia persica group (Figure 12). Otostegia persica preserved the normal morphology of the kidney after skeletal muscle ischemia-reperfusion.
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Histological examination confirmed the extent of renal changes in the ischemia-reperfusion group was
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higher than other groups.
4. Discussion
Ischemia-reperfusion in the lower extremities is one of the most common kinds of injuries that occur in various conditions. It is clear that skeletal muscle is the tissue in a limb that is most vulnerable to ischemia. Because muscle represents the primary mass of tissue in the extremity, damage to muscle is the
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most critical aspect of the limb reperfusion syndrome.
During ischemia, muscle cells cannot keep their membrane integrity and this causes releasing of calcium, phospholipid A2, formation of polyunsaturated fatty acids and fatty acids radicals. If the
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oxygenation is re-established at that stage of ischemia, fatty acid radicals react with oxygen and undergo lipid peroxidation reaction. This reaction increases the membrane permeability and also stimulates chemotaxis of leukocytes, which release oxygen-derived free radicals and proteolytic enzymes when
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activated. Activated leukocytes release a variety of inflammatory mediators, including neutrophil proteases, cytokines and reactive oxygen species. All of these products cause damage to adjacent endothelial cells and they have been thought to play key roles in tissue injury. 28 For these reasons antioxidants are of interest for the treatment of ischemia-reperfusion injury. Restoring blood flow to the limbs exposed to ischemia can cause the reperfusion injury by leading to formation of free radicals that lead to more muscle necrosis than that caused by the ischemia itself.29,30 Reperfusion injury develops on remote organs such as lungs, liver, heart and kidneys that threatening life.31-33
ACCEPTED MANUSCRIPT Several experimental studies had model and design similar to our study to investigate the effectiveness of antioxidant drugs in kidney as a remote organ injury after skeletal muscle ischemia-reperfusion.
9,34-36
Our data, in concert with previous ones,8-12 confirmed that reperfusion of the ischemic limb leads, within 24h of reperfusion, to a systemic response as represented by the morphological and functional impairment
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of the kidney. Antioxidants are compounds that postpone or inhibit the oxidation process by blocking the initiation or diffusion of oxidizing chain reactions. There are two basic classes of antioxidant namely natural and synthetic ones. Limitation on the use of synthetic antioxidants is being imposed because of their
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carcinogenicity.37,38 Thus the concern in natural antioxidants has been increased extremely. As resources of natural antioxidants much consideration has been paid to plants.39,40 The antioxidant effect of several
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Iranian plants has been reported.41 Yassa et al13 reported that methanolic extract of the Otostegia persica has antioxidant properties. Hedayati et al15 showed that administering Otostegia persica extract for 3 and 6 days decreases glucose, creatinine, and even urea serum levels in the diabetic rats. The results of Hajhashemi et al42 study indicated that hydroalcoholic extract of Otostegia persica has component that could alleviate the morphine withdrawal syndrome. Also the methanol extract of Otostegia persica has
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protective effect against the liver damage and hepatoprotective mechanisms of this extract on Carbon Tetrachloride-induced liver damage might be due to the decreased lipid peroxidation (decreased MDA level and increased content of GSH).19 The therapeutic effect of Otostegia persica in ischemia and its role
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in prevention of reperfusion has not been addressed yet. The absence of studies, using Otostegia persica in renal remote injury after skeletal muscle ischemia-reperfusion, we have motivated the design of this research. In this study it was hypothesized that Otostegia persica might have efficacy in treating ischemia
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and would decline the metabolic syndrome of reperfusion protects target and remote organs. The results of our study confirm that Otostegia persica extraction administration could significantly decrease the serum creatinine and urea levels, water intake, urinary excretion rate, ratio of kidney/body weight, renal tissue malondialdehyde values, enzymatic activity of catalase and superoxide dismutase, glutathione level and preserve the normal morphology of the kidney after skeletal muscle ischemiareperfusion. These results suggest that Otostegia persica extraction pretreatment could have a positive effect to reduce renal remote injury.
ACCEPTED MANUSCRIPT 5. Conclusion Our data support a role for Otostegia persica in attenuation of renal remote injury after skeletal muscle ischemia-reperfusion in an animal model, in part at least by antioxidant or free radical scavenging
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activity.
References
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ACCEPTED MANUSCRIPT 24. Ashtiyani SC, Najafi H, Kabirinia K et al. Oral Omega-3 fatty acid for reduction of kidney dysfunction induced by reperfusion injury in rats. Iran J Kidney Dis 2012; 6(4) 275-83. 25. Claiborne A. CRC handbook of methods for oxygen radical research. Boca Raton: CRC Press; 1985. 26. Kono Y. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for
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ACCEPTED MANUSCRIPT 38. Ito N, Fukushima S, Hasegawa A et al. Carcinogenicity of butylated hydroxyl anizole in F344 rats. J Natl Cancer Inst 1983; 70: 343-4. 39. Couladis M, Tzakou O, Verykokidou E. Screening of some Greek aromatic plants for antioxidant activity. J Phytother Res 2003; 17: 194-6.
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42. Hajhashemi V, Rabbani M, Asghari GR et al. Effects of Otostegia persica (Burm.) Boiss on morphine
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withdrawal syndrome in mice. Iranian Journal of Pharmaceutical Research 2004; 3: 171- 5.
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Table 1
Sham
-
+ -
Otostegia persica -
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Tubular epithelial necrosis score Polymorphonucleocyte in outer medulla Cellular vacuolation Interstitial edema Tubular dilation Hyaline cast Medullary congestion
Control
Ischemiareperfusion +++ ++ +++ ++ ++ +++ +
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Feature
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Histopathological changes of kidneys in rats. Ischemia-reperfusion + Otostegia persica + + + -
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ACCEPTED MANUSCRIPT
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FIG. 1. Effect of Otostegia persica (OP) on serum creatinine. Values are expressed as mean ± standard deviation and one way ANOVA with Bonferroni test was performed. ***: P
S1743-9191(14)01010-3
DOI:
10.1016/j.ijsu.2014.11.047
Reference:
IJSU 1673
To appear in:
International Journal of Surgery
Received Date: 1 March 2014 Revised Date:
24 November 2014
Accepted Date: 29 November 2014
Please cite this article as: Takhtfooladi MA, Takhtfooladi HA, Moayer F, Karimi P, Asl HA, Effect of Otostegia Persica extraction on renal injury induced by hindlimb ischemia-reperfusion: a rat model, International Journal of Surgery (2015), doi: 10.1016/j.ijsu.2014.11.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Effect of Otostegia Persica extraction on renal injury induced by hindlimb ischemia-
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reperfusion: a rat model
Mohammad Ashrafzadeh Takhtfooladi1, Hamed Ashrafzadeh Takhtfooladi2, Fariborz Moayer3, Poorya Karimi4,
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Hesam Abbasian Asl4
1
Young Researchers and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2
Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
3
Department of Pathobiology, College of Veterinary Medicine, Karaj Branch, Islamic Azad University, Alborz, Iran.
4
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Department of Clinical Sciences, College of Veterinary Medicine, Karaj Branch, Islamic Azad University, Alborz, Iran.
Correspondence to: Mohammad Ashrafzadeh Takhtfooladi. Young Researchers and Elites Club, Science and
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Research Branch, Islamic Azad University, Tehran, Iran. Tel. 00989121590428. Fax: 00983117860211. E-mail:
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[email protected]
ACCEPTED MANUSCRIPT ABSTRACT Introduction: It is known that ischemia-reperfusion causes remote organ injury as well as local injury. In traditional systems of medicine, many plants have been documented to be useful for the treatment of various disorders including oxidative esters. This study was designed to investigate whether Otostegia
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persica extraction pretreatment has a protective effect against renal injury induced by hindlimb ischemiareperfusion. Methods: Forty male Wistar rats were allocated into five groups as follows: Control, Sham, Otostegia persica, ischemia-reperfusion and ischemia-reperfusion + Otostegia persica groups. Rats in Otostegia persica and ischemia-reperfusion + Otostegia persica groups received Otostegia persica
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extraction (300 mg/kg) orally 2 days prior to operation. Hindlimb ischemia was induced by clamping the femoral artery for 2 hours. After 24 hours of reperfusion, blood and urine samples were obtained for
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kidney function tests and the kidneys were removed for histological analysis and oxidative stress measurement. Results: The decrease in glomerular filtration rate induced by reperfusion was significantly improved by Otostegia persica extraction administration (P < 0.05), which resulted in the decrease in serum urea and creatinine concentrations. Urinary creatinine significantly decreased in ischemiareperfusion group compared to the other groups (P < 0.05). Urinary excretion rate, water intake and the
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ratio of kidney/body weight significantly increased in animals with reperfusion injury as compared with other groups (P < 0.05). On histological examination, rats pretreated with Otostegia persica extraction had nearly normal morphology. Skeletal muscle ischemia-reperfusion produced a significant increase in
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renal tissue malondialdehyde level, while pretreatment with Otostegia persica extraction was associated with a significantly lower malondialdehyde level (P < 0.05). Renal tissue catalase and superoxide dismutase activity and glutathione level were significantly (P < 0.05) decreased by hindlimb ischemia-
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reperfusion. The increases in these parameters were decreased by pretreatment with Otostegia persica extraction. Conclusions: The results of this study showed that Otostegia persica extraction pretreatment significantly protected the renal injury from skeletal muscle ischemia-reperfusion.
Keywords: skeletal muscle; ischemia-reperfusion; remote organ damage; kidney injury; Otostegia persica.
ACCEPTED MANUSCRIPT 1. Introduction Skeletal muscle ischemia-reperfusion injury is common in several clinical practice including trauma, disease and orthopedic surgery.1 Reperfusion injury of the extremities is characterized by metabolic acidosis, increased serum creatine kinase, myoglobinury, hyperkalemia by the loss of intracellular
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potassium and free radicals release.2 Numerous free radicals are brought along when oxygenated blood flow re-enters ischemic tissues during reperfusion, leading to additional tissue injury.3
The development of remote organ dysfunction was observed only following reperfusion, which implies that cellular mediators produced locally in the limb were responsible for mediating remote organ
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injury.4,5 Histological and biochemical evidences of lung, liver and kidney dysfunction indicates that multiple organ dysfunctions following hindlimb ischemia-reperfusion occur as a central systemic event
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rather than sequential failure of individual organs.5-7
Multiple pharmacological agents such as iloprost,8 vitamin C,8 pentoxifylline,9 L-alanyl-glutamin,10 N-acetylcysteine11 and erythropoietin12 are proposed to be useful against renal remote injury after hindlimb ischemia-reperfusion.
Otostegia persica (Burm.) Boiss., that locally known as “Golder” is an endemic medicinal plant
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growing in southern and also southeastern of Iran.13 The extract of the Otostegia persica is used in Iranian traditional medicine as anti-histaminic, anti-spasmodic, anti-arthritis, anti-pyretic and analgesic in toothache.14,15 There are evidences that Otostegia persica extraction also is an effective treatment for saving the burn site.16 The aerial part of Otostegia persica is reported to have high antioxidant activity
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which is related to the flavonoids.17 Additional biological screening of the methanolic extract has revealed strong antioxidant as well as antibacterial activities against various strains of Gram-negative and Gram-
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positive bacteria.17,18 According to experiments, it is determined that methanolic extract of the Otostegia persica has antioxidant properties.13 Despite many uses of Otostegia persica, no data can be found about the antioxidant role of the plant in reperfusion syndrome. This prompted us to carry out an investigation for effects of Otostegia persica on renal remote organ injury after hindlimb ischemia-reperfusion in rat model.
2. Material and methods
ACCEPTED MANUSCRIPT All experimental procedures were performed according to the guidelines for the ethical treatment of experimental animals and approved by Islamic Azad University School of Veterinary Science, Animal Care and Use Local Ethics Committee.
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2.1. Experimental protocol The sample size is calculated using the following formula:
where n is the required sample size. For Z α, Z is a constant (set by convention according to the accepted α
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error and whether it is a one-sided or two-sided effect) as shown below:
For Z1-β, Z is a constant set by convention according to power of the study as shown below:
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In the above-mentioned formula σ is the standard deviation (estimated) and ∆ the difference in effect of two interventions which is required (estimated effect size). Forty male Wistar rats weighing 300±35g were used in this study. All rats were maintained under
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constant room temperature of 25 ± 2°C, 12h/12h light/dark cycle with access to water and commercial food ad libitum and placed in individual cages. Animals were randomly allocated into five experimental
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groups of eight rats each:
Group I - Control group, with no operation and no treatment. Group II - Sham group with no ischemia-reperfusion. The animals were subjected to all operative procedures (isolation and exposure of the femoral artery for 2h) except arterial occlusion and reperfusion. Group III (Otostegia persica group) - which only received orally Otostegia persica extraction (300 mg/Kg)19 by using gavage technique, 2 days prior to operation. Group IV (ischemia-reperfusion group) - The animals were subjected to ischemia-reperfusion. Group V (ischemia-reperfusion+Otostegia persica group) - Otostegia persica extraction, 300 mg/kg, was orally administered 2 days prior to induction of ischemia-reperfusion.
ACCEPTED MANUSCRIPT Anesthesia was induced using intramuscular ketamine hydrochloride 10% (50 mg/kg) plus xylazine hydrochloride 2% (10 mg/kg). After the induction of anesthesia, 250 IU heparin was administered via the jugular vein to prevent clotting. Then, a skin incision (by using a sterile technique) was made on the medial surface of the left hindlimb. After the isolation of the femoral artery and vein from the
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surrounding tissues, the femoral artery was exposed. Hindlimb ischemia was induced by clamping the femoral artery. Rats were maintained in dorsal recumbency and kept anesthetized (additional doses were given as necessary) throughout the duration of the ischemic period. Body temperature was maintained with a heating pad and monitored using a rectal thermometer. After 2h ischemia, the vascular forceps was
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removed and surgical site was routinely closed. After surgery, fluid losses were replaced by administration of 5ml of warm (37˚C) isotonic saline i.p, and rats were kept in metabolic cages for 24h to
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collect urine and also to measure water consumption.
2.2 Plant material
The plant Otostegia persica was collected from Kerman province, Iran. The plant was identified by the Department of Botany of the Islamic Azad University. A voucher specimen has been deposited at the
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Herbarium of Department of Botany. Aerial parts of Otostegia persica were finely powdered in a mill. 500g of sample was selected and raised the volume to 1L by ethanol (96%). The solution percolated after 48h, then the solvent was removed under reduced pressure at low temperature and finally about 10g
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concentrated extract was prepared. Doses of the extract were prepared using normal saline.14,20
2.3. Biochemical analysis
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After reperfusion period, the blood samples were collected from jugular vein and were allowed to clot for 10 min at room temperature. Clots were centrifuged at 2500 rpm for 10 min to separate the serum and submitted for evaluation of serum creatinine and urea levels. Creatinine and urea concentrations of serum and urine creatinine were measured spectrophotometrically by using commercial kits by Automatic DADE AnalyzerTM.
2.4. Histological analysis
ACCEPTED MANUSCRIPT After euthanizing the animals by overdose of intraperitoneal pentobarbital injection (300 mg/kg), their kidneys were quickly removed. The kidneys from each rat were weighted for measurement of the ratio of kidney/body weight and the left kidneys were used for histological analysis under light microscopy. Renal tissues were placed in 10% formalin solution and processed according to standard procedures by
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embedding in paraffin then tissues were sectioned in 5µm pieces and stained with Hematoxylin-Eosin stain. Specimens were evaluated by one pathologist who was blinded to the experiment and data. All specimens were examined for 7 histological parameters including cellular vacuolation, apoptosis, interstitial edema, tubular dilatation, hyaline cast, polymorphonucleocytes in outer medulla, and
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medullary congestion on a semiquantitative scale of none (–), mild (+), moderate (++), and severe (+++).21 Tubular epithelial necrosis was graded as follow: normal histology (–), tubular cell swelling and
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nuclear condensation with up 1/3 of tubular profile exhibiting nuclear loss (+),tubular cell swelling and nuclear condensation with 1/3 to 2/3 of tubular profile exhibiting nuclear loss (++), greater than 2/3 of tubular profile showing nuclear loss (+++).21
2.5. Preparation of renal tissue homogenates
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Renal tissue homogenate and supernatant samples were prepared as described by Bhalodia et al.22 In brief, right kidneys perfused immediately with icecold hypertonic saline solution, and homogenized in chilled potassium chloride (1.17%). The homogenate was centrifuged at 10500g for 20 minutes at 4˚C to
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get the postmitochondrial supernatant, which was used to assay lipid peroxidation activity. 22
2.6. Measurement of lipid peroxidation
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For evaluating the status of oxidative stress, malondialdehyde (end product of lipid peroxidation) levels in renal tissue samples were measured. Malondialdehyde content in the renal tissue were measured by the method described by Ohkawa and colleagues.23 This is based on the measurement of thiobarbituric acid malondialdehyde absorbance. The reaction mixture consisted of 0.2 mL of 8.1% sodium lauryl sulphate, 1.5 mL of 20% acetic acid solution adjusted to a pH of 3.5 with sodium hydroxide, and 1.5 mL of 0.8% aqueous solution of thiobarbituric acid added to 0.2 mL of 10% (w/v) of postmitochondrial supernatant. The mixture was made up to 4.0 mL with distilled water and heated at 95°C for 60 minutes. After cooling with water, 1.0 mL of distilled water and 5.0 mL of the mixture of n-butanol: pyridine (15:1
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2.7. Measurement of antioxidant enzymes
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Catalase activity was measured by the method that described by Claiborne.25 The rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm. Superoxide dismutase activity was recognized according to the method of Kono.26 Nitro blue tetrazolium decline to blue formazan by superoxide anion was traced at 560 nm. Non-protein sulfhydryl, as a marker
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at 412 nm according to the method of Jollow et al.27
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for reduced glutathione, was measured by the reduction of Ellman’s reagent by -SH groups that was read
2.8. Statistics
Statistics were performed on SPSS software (version 18). Data were expressed as the mean±standard deviation and analyzed by one way ANOVA, with the significance of individual comparisons being assessed by Bonferroni test, and unpaired Student’s t-test was performed where appropriate. Differences
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were considered significant at P values less than 0.05.
3. Results
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During the study, two animals died; one rat from group II during anesthesia, and one rat from group IV in the following first hour after surgery (all were replaced immediately). There were no symptoms
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before death; dead rats underwent necropsy without significant findings.
3.1. Biochemical findings
Rats that underwent skeletal muscle ischemia-reperfusion exhibited significant increase in serum concentrations of urea and creatinine levels as compared with other groups (P < 0.05). Pretreatment with Otostegia persica extraction significantly decreased serum creatinine and urea levels compared with the ischemia-reperfusion group (P < 0.05) (Figures 1,2). In groups with reperfusion induction, urinary creatinine significantly decreased compared to other groups (P < 0.05) (Figure 3).
ACCEPTED MANUSCRIPT 3.2. Urinary excretion, water intake and kidney/body weight ratio Urinary excretion rate increased in rats with reperfusion injury as compared with the other rats (P < 0.05). It significantly decreased by pretreatment of Otostegia persica extraction in ischemia-reperfusion + Otostegia persica group (P < 0.05) (Figure 4). Water intake increased in animals with reperfusion injury
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as compared with rats in the ischemia-reperfusion + Otostegia persica group (P < 0.05), control and sham animals, and it significantly decreased by pretreatment of Otostegia persica extraction in the ischemiareperfusion + Otostegia persica group (P < 0.05) (Figure 5). Eventually, the ratio of kidney weight to body weight also increased in the ischemia-reperfusion group as compared with Otostegia persica,
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control and sham groups (P < 0.05). Pretreatment of Otostegia persica extraction decreased this ratio
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compared with no Otostegia persica extraction in the ischemia-reperfusion group (p < 0.05) (Figure 6).
3.3. Lipid peroxidation activity
Figure 7 illustrates that the malondialdehyde values of kidney tissues in the sham and Otostegia persica groups were not significantly different (P > 0.05); however, occlusion of the femoral artery for 2h and reperfusion for 24h increased the level of renal tissue malondialdehyde in the ischemia-reperfusion
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group in comparison with the sham group (P < 0.05). Otostegia persica extraction administration could significantly decrease the malondialdehyde values in the ischemia-reperfusion + Otostegia persica group compared with its corresponding values in the ischemia-reperfusion group (P < 0.05), yet it was still
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significantly different from the sham group (P < 0.05).
3.4. Antioxidant activity
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Rats in the ischemia-reperfusion group showed significantly decreased enzymatic activity of catalase (P < 0.05) (Figure 8), superoxide dismutase (P < 0.05) (Figure 9), and reduced glutathione (P < 0.05) (Figure 10) when compared with the other groups rats. These declining trends were significantly less in the group that received Otostegia persica extraction compared with those in the ischemia-reperfusion group.
3.5. Histological findings
ACCEPTED MANUSCRIPT The histological changes were summarized in the Table 1. The sham and Otostegia persica groups of rats did not show any renal morphological changes. By contrast, the kidneys of the rats with ischemiareperfusion showed significant pathologic changes of tubular cell swelling, tubular dilatation, interstitial edema, moderate to severe necrosis, cast formation in tubule and glomerul, glomerul fibrosis and
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hemorrhage (Figure 11). Apoptotic renal epithelial cells and nuclear changes consisting of karyolysis, picnosis, and karyorhexia were also notified in the ischemia-reperfusion group. Slight histological alterations were seen in the ischemia-reperfusion + Otostegia persica group (Figure 12). Otostegia persica preserved the normal morphology of the kidney after skeletal muscle ischemia-reperfusion.
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Histological examination confirmed the extent of renal changes in the ischemia-reperfusion group was
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higher than other groups.
4. Discussion
Ischemia-reperfusion in the lower extremities is one of the most common kinds of injuries that occur in various conditions. It is clear that skeletal muscle is the tissue in a limb that is most vulnerable to ischemia. Because muscle represents the primary mass of tissue in the extremity, damage to muscle is the
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most critical aspect of the limb reperfusion syndrome.
During ischemia, muscle cells cannot keep their membrane integrity and this causes releasing of calcium, phospholipid A2, formation of polyunsaturated fatty acids and fatty acids radicals. If the
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oxygenation is re-established at that stage of ischemia, fatty acid radicals react with oxygen and undergo lipid peroxidation reaction. This reaction increases the membrane permeability and also stimulates chemotaxis of leukocytes, which release oxygen-derived free radicals and proteolytic enzymes when
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activated. Activated leukocytes release a variety of inflammatory mediators, including neutrophil proteases, cytokines and reactive oxygen species. All of these products cause damage to adjacent endothelial cells and they have been thought to play key roles in tissue injury. 28 For these reasons antioxidants are of interest for the treatment of ischemia-reperfusion injury. Restoring blood flow to the limbs exposed to ischemia can cause the reperfusion injury by leading to formation of free radicals that lead to more muscle necrosis than that caused by the ischemia itself.29,30 Reperfusion injury develops on remote organs such as lungs, liver, heart and kidneys that threatening life.31-33
ACCEPTED MANUSCRIPT Several experimental studies had model and design similar to our study to investigate the effectiveness of antioxidant drugs in kidney as a remote organ injury after skeletal muscle ischemia-reperfusion.
9,34-36
Our data, in concert with previous ones,8-12 confirmed that reperfusion of the ischemic limb leads, within 24h of reperfusion, to a systemic response as represented by the morphological and functional impairment
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of the kidney. Antioxidants are compounds that postpone or inhibit the oxidation process by blocking the initiation or diffusion of oxidizing chain reactions. There are two basic classes of antioxidant namely natural and synthetic ones. Limitation on the use of synthetic antioxidants is being imposed because of their
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carcinogenicity.37,38 Thus the concern in natural antioxidants has been increased extremely. As resources of natural antioxidants much consideration has been paid to plants.39,40 The antioxidant effect of several
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Iranian plants has been reported.41 Yassa et al13 reported that methanolic extract of the Otostegia persica has antioxidant properties. Hedayati et al15 showed that administering Otostegia persica extract for 3 and 6 days decreases glucose, creatinine, and even urea serum levels in the diabetic rats. The results of Hajhashemi et al42 study indicated that hydroalcoholic extract of Otostegia persica has component that could alleviate the morphine withdrawal syndrome. Also the methanol extract of Otostegia persica has
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protective effect against the liver damage and hepatoprotective mechanisms of this extract on Carbon Tetrachloride-induced liver damage might be due to the decreased lipid peroxidation (decreased MDA level and increased content of GSH).19 The therapeutic effect of Otostegia persica in ischemia and its role
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in prevention of reperfusion has not been addressed yet. The absence of studies, using Otostegia persica in renal remote injury after skeletal muscle ischemia-reperfusion, we have motivated the design of this research. In this study it was hypothesized that Otostegia persica might have efficacy in treating ischemia
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and would decline the metabolic syndrome of reperfusion protects target and remote organs. The results of our study confirm that Otostegia persica extraction administration could significantly decrease the serum creatinine and urea levels, water intake, urinary excretion rate, ratio of kidney/body weight, renal tissue malondialdehyde values, enzymatic activity of catalase and superoxide dismutase, glutathione level and preserve the normal morphology of the kidney after skeletal muscle ischemiareperfusion. These results suggest that Otostegia persica extraction pretreatment could have a positive effect to reduce renal remote injury.
ACCEPTED MANUSCRIPT 5. Conclusion Our data support a role for Otostegia persica in attenuation of renal remote injury after skeletal muscle ischemia-reperfusion in an animal model, in part at least by antioxidant or free radical scavenging
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activity.
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withdrawal syndrome in mice. Iranian Journal of Pharmaceutical Research 2004; 3: 171- 5.
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Table 1
Sham
-
+ -
Otostegia persica -
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Tubular epithelial necrosis score Polymorphonucleocyte in outer medulla Cellular vacuolation Interstitial edema Tubular dilation Hyaline cast Medullary congestion
Control
Ischemiareperfusion +++ ++ +++ ++ ++ +++ +
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Feature
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Histopathological changes of kidneys in rats. Ischemia-reperfusion + Otostegia persica + + + -
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FIG. 1. Effect of Otostegia persica (OP) on serum creatinine. Values are expressed as mean ± standard deviation and one way ANOVA with Bonferroni test was performed. ***: P
