Effect of Ellagic Acid, Cilostazol and Their Combination on Amikacin Induced Nephrotoxicity in Rats

Amikacin(AK) has the largest spectrum of aminoglycosides. However, its use is limited due to nephrotoxicity and ototoxicity. Ellagic acid (EA) is a plant phenolic structure. it has antioxidant, anticarcinogenic and antimutagenic properities. Cilostazol (CTZ) is a PDE Ш inhibitor, it is a potent vasodilator and antiplatelet drug. This study aimed to determine if EA and cilostazol have a protective effect against nephrotoxicity caused AK. Forty nine rats were divided into seven equal groups: control normal; AK 400mg/kg; EA 10 mg/kg; CTZ 10mg/kg; AK 400mg/kg plus EA 10mg/kg; AK 400mg/kg plus CTZ 10mg/kg; AK 400mg/kg plus EA 10mg/kg and CTZ 10mg/kg. For seven days, Drugs were given orally one hour before intramuscular injection of AK. After twenty-four hours from the last dosage, samples of blood were obtained to determine blood urea nitrogen (BUN) and creatinine levels in serum, kidneys were extracted and longitudinally divided into two parts, part for measuring the following parameters: malondialdehyde (MDA), catalase (CAT), reduced glutathione (GSH), interleukin 6 (IL6), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNFα), nuclear factor kappa B (NFκB) and Bcl-2 associated x protein (Bax), the other part was placed in formaldehyde solution and examined under light microscopy for routine histopathologic examination. The results of the present study proved that EA, CTZ and their combination protected rats against AK - induced nephrotoxicity; This effect might be a result of the antioxidant, anti-inammatory and anti-apoptotic properties of these compounds. Blood samples from the retro-orbital plexus of collected microcapillary centrifugation 3000 × g 10 to separate serum for determining serum BUN and creatinine concentrations on the 8th histopathological examination, left kidney longitudinal section designated. Additionally, biochemical estimation of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), nuclear factor


Introduction
Amikacin (AK) has the broadest spectrum and the least resistance of all aminoglycosides. AK is preferred due to its advantageous characteristics, which include rapid and robust bactericidal activity, synergy with β-lactam antibiotics, low cost, chemical stability and low resistance; however, its use is limited due to the risk of nephrotoxicity and ototoxicity (Abdel-Daim et al. 2019).
Because AK is not metabolized in the body and is eliminated in large amounts in the urine, it builds up in the proximal convoluted tubules, causing free radical production. Nephrotoxicity is caused by these free radicals (Ozer et al. 2020).
Multiple mechanisms, such as in ammation, blockage of particular transporters, production of oxidative stress and decreased renal blood ow are involved in amikacin-induced renal damage (Prajapati and Singha 2010).
Ellagic acid (EA) is a polyphenolic compound found in plants naturally. Several studies have shown that EA has antioxidant, anti-apoptotic and anticarcinogenic properties. This antioxidant action of this compound is determined by its chemical structure, speci cally the number of hydroxyl groups and their ability to boost the stability of the phenoxyl radical (Firdaus et al. 2018).
EA decreases the expression of proin ammatory and pro brogenic cytokines such as tumor necrosis factor-alpha (TNFα), tumor growth factor-alpha (TGFα), and many interleukins which are involved in alcohol-induced in ammation and brosis (Ciuclan et al. 2010).
Cilostazol (CTZ) is a strong antiplatelet and vasodilator that is a speci c PDE III inhibitor. It raises intracellular cyclic adenosine monophosphate(cAMP) levels (Ragab et al. 2014). It also raises cyclic guanosine monophosphate (cGMP),it has many effects in different tissues (Rondina and Weyrich 2012).
Quantitative estimation of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL6) concentration in renal tissue: It was assessed using USCN life Science Inc. ELISA kits. The competitive inhibition enzyme immunoassay technique is used in this assay.
Estimation Bcl-2 associated x protein (Bax) and nuclear factor kappa B (NFκB) in renal tissue: It was determined using quantitative real-time PCR after total RNA was isolated according to the manufacturer's instructions using the Qiagen tissue extraction kit (Qiagen, USA). Using a high-capacity cDNA reverse transcription kit (Fermentas, USA), total RNA was converted to cDNA. Using Applied Biosystems with Step One TM software version 3.1 (USA), ampli cation and analysis of the real-time qPCR product were performed. The primer sequence of the studied genes; BAX: Forward primer:5'-CCCTGTGCACTAAAGTGCCC-3. Reverse primer: 5'-CTTCTTCACGATGGTGAGCG-3 NFκB: Forward primer:5'-CATTGAGGTGTATTTCACGG -3 Reverse primer: 5'-GGCAAGTGGCCATTGTGTTC -3 Histopathological studies: Prior to sacri ce, the animals were anesthetized with a 50 mg/kg intraperitoneal injection of sodium pentobarbital.
Both kidneys were quickly removed and opened. The specimens were xed in 10% formalin, sectioned into 5mm thick para n blocks and hematoxylin and eosin stains (H&E) were used for light microscopy (Bancroft and Gamble 2002) Analysis of data:. To compare all groups, one-way analysis of variance (ANOVA) was performed, while to compare between every two groups the least signi cant difference (LSD) was utilized. All data are expressed as mean ± SEM. A P-value less than 0.05 is considered signi cant. Computer analysis of the collected data was performed using the Statistical Package for Social Services version 25 (SPSS).

Results
Effect on BUN and Creatinine: AK 400mg/kg signi cantly increased BUN and creatinine levels in comparison to the control normal group. EA 10mg/kg or CTZ 10mg/kg alone produced a non signi cant reduction in both parameters in relation to the control normal group. AK plus EA and AK plus CTZ produced a signi cant reduction of BUN and creatinine compared with AK group. AK plus EA and CTZ produced more signi cant reduction of both parameters than each drug alone (Table 1).
Effect on MDA, GSH, SOD and CAT: AK 400mg/kg signi cantly increased MDA levels in renal tissue and caused a signi cant reduction of CAT, SOD, and GSH in renal tissue compared to the control group. EA10mg/kg or CTZ 10mg/kg alone produced non signi cant results in relation to the control normal group. AK plus EA and AK plus CTZ signi cantly reduced MDA and signi cantly increased GSH, SOD and CAT in renal tissue in relation to the AK group. AK plus EA and CTZ produced more signi cant reduction of MDA and more signi cant rise of GSH, SOD and CAT than each drug alone ( Table 2).
Effect on TNFα and IL6 AK 400mg/kg produced a signi cant increase in renal tissue TNFα and IL6 as compared to the control normal group. EA 10mg/kg or CTZ 10mg/kg alone produced non signi cant results in relation to the control group. AK plus EA and AK plus CTZ produced a signi cant reduction of TNFα and IL6 in renal tissue as compared to the AK group. AK plus EA and CTZ produced more signi cant reduction of TNFα and IL6 than each drug alone (Table 3).
Effect on expression of NFκB and Bax AK 400mg/kg resulted in a signi cant increase of NFκB and BAX expression in renal tissue compared with the control normal group. EA 10mg/kg or CTZ 10mg/kg alone produced a non signi cant results in relation to the control normal group, while AK plus EA and AK plus CTZ showed a signi cant decrease of NFκB and BAX expression in relation to AK group. AK plus EA and CTZ produced more signi cant reduction of NFκB and BAX expression in renal tissue than each drug alone (Table 4).  In this study, oral adminsteration of EA 10mg/kg and CTZ 10mg/kg before AK 400mg/kg produced more reduction of BUN and creatinine than each drug alone due to the additive effect of both drugs. The ability of EA to scavenge free radicals has been related to its intrinsic antioxidant activity. This is due to the fact that it can transfer the phenolic H-atom to a free radical. Lactone systems and EA hydroxyl groups can create Hydrogen bonds, and they can act as hydrogen donors and electron acceptors. As a result, EA possesses ability to participate in antioxidant redox reactions, resulting in a highly e cient free radical scavenger (Ríos et al. 2018).
Oxidative stress has been demonstrated to be reduced by EA through modulation of multiple mechanisms. These In this present work, EA 10mg/kg orally one hour before AK signi cantly reduced TNFα, IL6 and NFκB expression. Aminoglycosides can cause apoptosis in the kidney by increasing the content of cytosolic Bax protein, which leads to activating the mitochondrial pathway of apoptosis, it includes caspase 9 activation as an initiator, caspase 3 activation as an effector and DNase activation, resulting in DNA fragmentation and apoptosis (Servais et al. 2006).
In this study, EA 10mg/kg orally one hour before AK reduced the expression of BAX in renal tissue. Sepand et al. This study proved that the combined effect of EA 10mg/kg plus CTZ 10mg/kg before AK produced more signi cant reduction of BAX expression in renal tissue than each drug alone which may occur may be due to the additive effect of both drugs.
The current histopathology ndings revealed that AK 400mg/kg was associated with disturbances in the kidney histopathological picture, including in ammatory cell in ltration, tubular epithelial lining degeneration and tubular

Declarations
Author contributions MK and NK conceived and designed the research. ZS and AS conducted experiments. ZS and AS analyzed the data. ZS wrote the manuscript. All authors read and approved the manuscript and all data were generated inhouse and that no paper mill was used.
Compliance with ethical standards The study was approved by Zagazig University's local animal Ethical Committee in Egypt. Approval number is ZU-IACUC/3/F/57/2019. National Institutes of Health's guidelines (USA) were followed all over the experiment.

Con ict of interest
The author declares no con ict of interest.
Funding statement