Protective Effects of DHA-PC against Vancomycin-Induced

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Protective Effects of DHA-PC against Vancomycin-Induced Nephrotoxicity through the Inhibition of Oxidative Stress and Apoptosis in BALB/c Mice Hao hao Shi, Jun zhe Zou, Tiantian Zhang, Hongxia Che, Xiang Gao, Cheng cheng Wang, YuMing Wang, and Changhu Xue J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04565 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 24, 2017

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Journal of Agricultural and Food Chemistry

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Protective Effects of DHA-PC against Vancomycin-Induced

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Nephrotoxicity through the Inhibition of Oxidative Stress and

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Apoptosis in BALB/c Mice

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Haohao Shi , Junzhe Zou , Tiantian Zhang , Hongxia Che , Xiang Gao§, Chengcheng Wang ,

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Yuming Wang

†#

‡#

†‖*

†

†

† ‖*

, Changhu Xue

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†

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Qingdao, Shandong Province 266003, PR China

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‡

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Road, Qingdao, Shandong Province 266100, PR China

College of Food Science and Engineering, Ocean University of China, No. 5 Yushan Road,

Teaching Center of Fundamental Courses, Ocean University of China, No. 238 Songling

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§

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Province 266071, PR China

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†

‖

College of Life Sciences, Qingdao University, No. 308, Ningxia Road, Qingdao, Shandong

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine

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Science and Technology, Qingdao, Shandong Province 266237, PR China

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# Haohao Shi and Junzhe Zou contributed equally to this work

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*Corresponding author:

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* Yuming Wang, College of Food Science and Engineering, Ocean University of China, No. 5

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Yushan Road, Qingdao, Shandong Province 266003, PR China

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Tel: 0532-82032597; Fax: 0532-82032597; E-mail: [email protected]

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* Changhu Xue, College of Food Science and Engineering, Ocean University of China, No. 5

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Yushan Road, Qingdao, Shandong Province 266003, PR China

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Tel: 0532-82032468; Fax: 0532-82032468; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The clinical use of glycopeptide antibiotic vancomycin is usually accompanied by nephrotoxicity,

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limiting its application and therapeutic efficiency. The aim of this study was to investigate the

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protection of DHA-enriched phosphatidylcholine (DHA-PC) against nephrotoxicity using a model

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of vancomycin-induced male BALB/c mice with renal injury by measuring death curves,

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histological changes and renal function indexes. The addition of DHA in DHA and DHA-PC

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groups were 300 mg/kg per day on the basis of human intake level in our study. Results indicated

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that DHA-PC could dramatically extend the survival time of mice, while traditional DHA and PC

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had no significant effects. Moreover, oral administration of DHA-PC exhibited better effects on

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reducing vancomycin-induced increases of blood urea nitrogen, creatinine, Cystatin C and kidney

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injury molecule-1 levels than traditional DHA and PC. DHA-PC significantly delayed the

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development of vancomycin-induced renal injury, including tubular necrosis, hyaline casts, and

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tubular degeneration. A further mechanistic study revealed that the protective effect of DHA-PC

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on vancomycin-mediated toxicity might be attributed to its ability to inhibit oxidative stress and

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inactivate mitogen-activated protein kinase (MAPK) signaling pathways, which was associated

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with upregulation of Bcl-2 and downregulation of Caspase-9, Caspase-3, Cytochrome-c, p38 and

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JNK. These findings suggest that DHA-PC may be acted as the dietary supplements or functional

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foods against vancomycin-induced nephrotoxicity.

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KEYWORDS

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vancomycin, nephrotoxicity, oxidative stress, apoptosis, DHA-PC

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INTRODUCTION It is generally accepted that vancomycin effectively inhibit the growth of methicillin-resistant

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Staphylococcus aureus (MRSA).

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limitations in its application and efficacy.4 Vancomycin-induced nephrotoxicity, a complex process,

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is involved with many factors and signaling pathways. In vancomycin-induced DNA damage,

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oxidative stress can affect intracellular signaling pathways such as mitogenactivated protein

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kinases (MAPKs), which may play an important role in acute kidney damage induced by

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vancomycin.5,

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reduction of vancomycin accumulation or activation, anti-oxidants,7 anti-inflammation or

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anti-apoptosis.5 Recently combination vancomycin with chemical composition of natural active

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substance or functional foods has drawn researchers’ great attention.8

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1, 2

However, its general side effect is nephrotoxicity,3 causing

There are various methods to prevent kidney injury mainly focusing on the

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A large number of studies have reported that phosphatidylcholine (PC) and fish oil might

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exhibit a beneficial protection against drug-induced kidney injury.9 PC from soy or egg exerted

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remarkable antioxidant activities and cytoprotection in various pathological processes including

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cisplatin-induced nephrotoxicity.10,

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nephrotoxicity remains unclear. In addition, polyunsaturated omega-3 fatty acids (n3-PUFAs),

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such as docosahexaenoic acid (DHA) from fish oil, have been proved to exhibit protection against

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many disorders including atherosclerosis, diabetes, cancers, inflammatory and immune renal

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diseases.12 Previous study showed that fish oil (300mg/kg) could improve the nephrotoxicity

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induced by gentamicin, cisplatin and adriamycin in rats possibly through intrinsic biochemical

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characteristics and antioxidant properties.13-17 However, little attention has been paid on fish oil to

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protect against kidney injury induced by vancomycin.5

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However, the protection of PC on vancomycin-induced

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In traditional fish oil, n3-PUFAs are mainly esterified to glycerol (TG) or ethyl ester (EE).

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Interestingly, n3-PUFAs in many marine materials are mainly present in the form of phospholipids

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(PLs), in which phosphatidylcholine is the main ingredient.18 DHA-PC are almost completely

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absorbed in the intestine and readily incorporated into cell membranes due to their unique

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molecular structure.19 Previous researches indicated that supplementation of DHA-PC was more

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effective than traditional DHA in alleviating MPTP-induced brain oxidative damage along with an 3



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improvement in motor function and metabolic parameters in mice fed with a high-fat die.20, 21

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Moreover, many studies suggested that DHA-PC had beneficial biological and nutritional

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functions including the ability to inhibit the neutrophil leukocyte-mediated inflammatory reaction

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and cancer growth, and reduce the individual’s blood lipid profile and the risk of cardiovascular

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disease.20 Furthermore, DHA-PC exhibited neuroprotective effects through antioxidant activity by

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inhibiting the mitochondria-dependent apoptotic pathway.20

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acute renal injury has still not been reported. Given ROS played a vital role in the pathological

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progression of acute kidney damage,

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vancomycin induced renal damage.

7

However, its protective effect in

so we hypothesized that DHA-PC could attenuate

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The purpose of the present research was to comparatively analyze the protection of DHA-PC,

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traditional DHA and PC on renal damage in vancomycin-induced mice by measuring death curves,

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histological changes and renal function indexes to demonstrate the underlying mechanisms.

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MATERIALS AND METHODS

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Chemicals and Reagents

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Vancomycin was purchased from Dalian Meilun Biotech Co., Ltd (Dalian, China); PC (the

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purity > 95%) from soybean was obtained from Avanti Polar Lipids, Inc. (Alabaster, Alabama,

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US); traditional DHA (the purity > 93%) from anchovy was purchased from Zhoushan Xinnuojia

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Co., Ltd. (Zhoushan, Zhejiang, China). Squid (Sthenoteuthis oualaniensis) roe was purchased

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from Weihai Boow Foods Co., Ltd. (Weihai, Shandong, China). Primary antibodies for Cyt-c,

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Bcl-2, Bax, Caspase-3, -9, p-P38, P38, p-JNK, JNK, β-actin as well as secondary antibodies were

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obtained from Cell Signaling Technology (Beverly, MA, USA). Creatinine(Cr), Blood urea

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nitrogen(BUN), Malondialdehyde (MDA), Catalase (CAT), Total antioxidant capacity (T-AOC),

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Glutathione (GSH) and Glutathione peroxidase (GPx) assay kits were obtained from Nanjing

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Jiancheng Bioengineering Institute(Nanjing, Jiangsu, China). The enzyme-linked immunosorbent

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assay kits for measuring Serum Cystatin C (Cys-C) and kidney injury molecule-1(KIM-1) levels

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were purchased from Wuhan USCN Business Co., Ltd (Wuhan, Hubei, China).

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Preparation of DHA-PC

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Total lipids were extracted from squid roe by the modified method as previous described.22, 23 4


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In brief, squid roe was smashed by homogenizer after lyophilize. The squid roe powder was

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extracted by chloroform and methanol solution at a volume ratio of 2:1 followed by mixing with

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NaCl solution (0.15 M) for 24 h, and then the chloroform was evaporated under vacuum to obtain

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the total lipids. The DHA-PC was separated from the above obtained total lipids by silica-gel

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column chromatography (20 × 1200mm, 200-300 meshes) eluted with chloroform (3L), acetone

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(0.5L), and methanol (2L), and DHA-PC was obtained after removing the methanol.21 The purity

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was 94% (Figure 1) determined by HPLC-ELSD (evaporate light-scattering detector) analysis

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using Agilent 1100 Series (Palo Alto, 27 CA, USA) packed with a silica normal-phase Zorbax

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Rx-SIL column (250 mm × 4.6 mm I.D., 5 mm, Agilent-Technologies, Palo Alto, CA, USA)

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eluted with a linear binary gradient using chloroform-methanol-25% ammonium hydroxide

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(80:19.5:0.5, v/v/v) and chloroform-methanol-25% ammonium hydroxide-water (60:34:0.5:5.5,

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v/v/v) at a flow rate of 1.0 mL/min according to the previous method.24 The chromatographic

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separation was carried out using. The fatty acid composition of DHA-PC was confirmed by gas

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chromatography packed with a HP-INNOWAX capillary column (30 m × 0.32 mm × 0.25 µm)

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using C15:0 as a standard fatty acid according to the previous method.20, 25 The lipids needed to be

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transmethylated by HCl/methanol (1:5) at 90 °C for 3 hours. The temperatures of the detector and

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injector were kept at 250 and 240 °C, respectively, and the oven temperature was increased from

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170 to 240 °C at 3 °C/min and kept at 240 °C for 15 min. Nitrogen was used as carrier gas (1.2

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mL/min). The contents of DHA and eicosapentaenoic acid (EPA) in DHA-PC were 41.91% and

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8.31%, respectively, as shown in Table 1.

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Experimental design

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Eight-week-old male Balb/c mice (20-22g) were acclimatized for 1 week. The protocols were

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approved by the ethical committee of experimental animal care at Ocean University of China

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(Qingdao, Shandong, China). Mice were randomly divided into 5 group including N (normal

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group); M (model group); PC (phosphatidylcholine group); DHA (traditional docosahexenoic acid

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group); DHA-PC (docosahexenoic acid-enriched phosphatidylcholine group), with each group

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comprising 17 animals. Except the normal group, vancomycin was injected intraperitoneally at a

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dose of 400 mg/kg every day, which could cause marked nephrotoxicity.26, 27 The mice were 5



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supplied with different samples by gavage for 4 days before injection of vancomycin, respectively.

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The addition of DHA in DHA and DHA-PC groups were 300 mg/kg per day on the basis of human

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intake level in our study. Notably, the DHA or PC content in these three samples treatment groups

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were basically the same.

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After treatment with vancomycin and samples for 4 days, 8 mice from each group were

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sacrificed for further evaluation, then blood and kidney tissues were collected. The serum was

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obtained by centrifuging blood at 4000 rpm for 15min and stored at -80℃ until use. The left

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kidney was weighed and stored at -80℃ until use. The right kidney was fixed in 10%

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formaldehyde for histopathological analysis. The remaining mice were continually received

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vancomycin and trial intervention, and their weighs were monitored every day. Finally, the death

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curves were drawn.

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Biochemical assays

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The levels of creatinine and blood urea nitrogen in the serum were evaluated by commercial

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assay kits based on the manufacturer's instructions. The evaluation of urine protein in urine was

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measured by the Coomassie brilliant blue method.

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The kidney tissue (100 mg) was homogenized in cold 0.1 M Tris-HCl buffer and the

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supernatant by centrifugation at 3000 rpm for 15 min was used for determining the levels of MDA,

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CAT, T-AOC, GSH and GPx by commercial assay kits based on the manufacturer's instructions.

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Histological analysis

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Tissues were processed with formalin and paraffin, and then cut about 5µm thick and stained

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with hematoxylin and eosin (H&E). Tubular damage was assessed by scoring tubular necrosis,

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epithelial damage, dilatation and cast formation in different fields under a microscope. Stained

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kidney sections were analyzed under a microscope using a 20× objective lens. A numeric grading

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scale from 0 to 4 was used to evaluate the changes of pathology including swelling, protein casts,

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and vacuolar degeneration. Grade 0, 1, 2, 3, 4 indicated no meaningful histopathological change,

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mild degree of change, mild multifocal pathology, moderate degree of change and severe

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pathology, respectively.

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Western blotting assay 6


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The kidney tissue (100 mg) was homogenized in RIPA lysis buffer supplemented with

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phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) and phosphatase inhibitor (Sangon

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Biotech Co., Ltd., Shanghai, China). Insoluble proteins were obtained by centrifugation at

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12000×g for 5 min at 4°C. Especially, the protein used for detecting Cyt-c was extracted from

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cytosol that was separated from mitochondria. Protein content was determined by BCA protein

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assay kits. Thirty micrograms protein was separated by SDS-polyacrylamide gel electrophoresis

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and the gel was transferred onto a PVDF membrane followed by incubating with blocking buffer

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for 2 h and primary antibodies of β-actin, Cyt-c, Caspases-3, Caspase-9, Bax overnight at 4°C.

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Protein bands were visualized by an enhanced chemiluminescence’s procedure after incubating

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with corresponding secondary antibodies for 2 h at room temperature. The scanned bands were

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determined by ImageJ software. The results were expressed as the relative values after

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normalization to β-actin.

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Statistical analysis

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Data were presented as the mean ± Standard error (SE). These data were evaluated using

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one-way ANOVA followed by Student’s t test and Tukey’s test using SPSS 18.0. These figures

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were carried out using software Prism 5.0 (GraphPad, San Diego, CA, USA). The difference was

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considered significant when p < 0.05.

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RESULTS AND DISCUSSION

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Survival curve of experimental mice

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In the present study, survival curve was used to evaluate the protective effects of traditional

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DHA, PC, and DHA-PC on vancomycin-induced nephrotoxicity and the results were shown in

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Fig2.28 All samples-pretreated groups and model group received continuously a dose of

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vancomycin (400 mg/kg body wt) and were followed up for 3wk. At 8d after injection of

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vancomycin, all of the mice in vancomycin group had expired. Interestingly, the survival rate of

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DHA-PC group was about 44.4% after 8d supplemental pretreatment, which exhibited the

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excellent protective effect among three samples-pretreated groups. However, the survival mice in

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traditional DHA group was only 11.1% and no mice was survival in PC group after 8d injection of

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vancomycin. Moreover, the survival time of DHA-PC-pretreated mice could be extended to 17 7



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days. The above results showed that pretreatment with DHA-PC improved survival rate after

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vancomycin administration in mice (Figure 2).

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Effects of different samples on metabolic parameters in mice

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The initial body weights of mice at 8 weeks of age were measured and no significant

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differences were observed. Metabolic parameters were determined after vancomycin treatment for

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4 days and the results were shown in Tables 2. After vancomycin administration for four days, the

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body weight and the food intake markedly decreased in vancomycin-treated mice compared with

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normal group. The kidney index of vancomycin-treated mice significantly increased by 43.0% in

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comparison with normal group. Treatment with all samples effectively attenuated the

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vancomycin-induced alterations in food intake, body weight and kidney-body weight ratio to some

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different degree. DHA-PC significantly blocked the significant decrease in the kidney index and

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body weight compared to vancomycin group. Food intake, body weight and kidney weight in the

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DHA group were slightly increased without statistical significance in comparison with

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vancomycin group. Administration of PC did not exhibit any alterations in these parameters

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without significant differences compared with vancomycin group. A large amount of research also

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suggested that the level of food intake, body weight change and kidney weight ratio could be

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markedly decreased in the mice which drug (cisplatin, patulin, gentamicin and ketorolac

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tromethamine)-induced acute kidney injury.12, 15, 29

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Urine volume and urine protein are marker indexes related to renal function. Proteinuria is

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the presence of excess proteins in the urine, and its excess could cause illness. In normal group,

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we only collected a trace of urine, and proteinuria level was slightest. However, there were

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significant increases of the urine volume and proteinuria levels in the vancomycin group. A

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significant reduce of urine volume and proteinuria levels was observed in the DHA-PC groups

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compared to the vancomycin group. The urine volume and proteinuria levels in the DHA group

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were slightly decreased when compared to vancomycin group.

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Histopathological results

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Kidney histological experiment was carried out to evaluate the protection of samples on

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vancomycin-induced nephrotoxicity. Previous studies suggested vancomycin also induced a 8


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functional change and histopathological damage in the kidney.30,

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histopathological results showed that vancomycin treatment caused several visible histology

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changes, including protein casts in renal tubules, swelling, vacuolization, proximal tubule necrosis,

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and tubular lumen, in which DHA-PC exhibited excellent protective effect against

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vancomycin-induced nephrotoxicity among the three samples groups. DHA treatment only could

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alleviate the vancomycin induced kidney damage to some extent. However, PC could not improve

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the vancomycin induced histological changes, and there were no significant differences between

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model group and PC-treated mice. Furthermore, in agreement with previous observations, DHA

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administration caused an improvement in renal function during antibiotics (gentamicin and

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tetrachlorodibenzo-p-dioxin)-induced nephrotoxicity.17

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Effects of different samples on vancomycin-induced kidney dysfunction

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As shown in Fig.3, the

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The levels of serum creatinine, BUN, Cys-C and KIM-1 have been considered as serum

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biochemical markers of renal damage.32 Serum creatinine, a byproduct of muscle metabolism, is

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excreted by the kidneys. Creatinine levels in blood and urine are usually used to determine the

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creatinine clearance, correlating approximately with the glomerular filtration rate (GFR). Blood

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urea nitrogen is the major end product of protein metabolism in human body and used as an

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indicator of glomerular filtration function. CysC is a cysteine protease inhibitor that is cleared

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only through glomerular filtration and is an ideal endogenous marker of changes in glomerular

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filtration rate. KIM-1 is renal proximal tubule epithelial cells in a transmembrane glycoprotein.

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In order to evaluate whether samples could relieve the renal injury in the vancomycin-treated

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mice, we determined the levels of creatinine, BUN, Cys-C and KIM-1 in serum. (Fig.4).

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Vancomycin administration caused a marked increase in creatinine, BUN, Cys-C and KIM-1

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levels compared with normal mice, suggesting a significant kidney injury. Oral treatment

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consecutively with DHA and DHA-PC for 4 days significantly reversed vancomycin-induced

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increases in creatinine, BUN, Cys-C and KIM-1 levels, especially DHA-PC showed a best effect

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in decreasing the creatinine level. Treatment with PC could only reduce the level of creatinine to

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some extent, whereas PC had no significant effects on serum levels of BUN, Cys-C and KIM-1

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(P>0.05). These results suggested that both DHA-PC and traditional DHA treatment protected 9



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vancomycin-induced nephrotoxicity in different degrees. Increasing recognition showed that

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received high doses vancomycin induced nephrotoxocity accompanied by ascended the levels of

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serum creatinine, BUN, Cys-C and KIM-1 in the mice. A previous study indicated that fish oil

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could protect the kidney from gentamicin-induced toxicity through decreasing the serum BUN and

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creatinine level.12 Moreover, Lee et al. found that PC might exhibit protection against

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cisplatin-induced renal injury in rats with reducing the creatinine level of serum.10 These

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experiments were similar with our results.

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Effects of different samples on vancomycin-induced mitochondria - mediated

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apoptosis

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Activation of caspases is a common event in apoptosis induction.33, 34 Caspase-3 is activated

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in the apoptosis both by extrinsic and intrinsic pathways.35, 36 Caspase-9 is an initiator caspase,

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encoded by the CASP9 gene which has been linked to the mitochondrial death pathway.

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Cytochrome-c plays an intermediate role in apoptosis, which is usually a controlled form of cell

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death in response to infection or DNA damage. Previous studies have demonstrated that caspase

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activation is involved in vancomycin-induced apoptosis.

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mechanisms contributing to the protection of samples on vancomycin-induced cytotoxicity, we

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measured the effects of PC, DHA and DHA-PC on caspase activation using Western blotting. As

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shown in figure 5, vancomycin alone significantly caused cleavages of caspase-3, -9, and

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cytochrome. These variations were significantly attenuated by sample. DHA-PC groups showed a

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significant reduction in caspase-3, -9 and cyt-c levels of kidney tissue compared to the

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vancomycin group. Notably, no significant differences were observed in the levels of cyt-c

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between the DHA-PC group and DHA group. Caspase-3, -9 and cyt-c levels in the PC group were

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slightly decreased when compared to the vancomycin group. Lu et al. also found that patulin

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caused nephrotoxicity with cleavages of caspase-3, -9.29

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To investigate the potential

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BCL-2 normally acted on the mitochondrial membrane to promote permeabilization and

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release of cytochrome C and ROS, playing a vital role in the promotion of cellular survival and

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inhibition of the pro-apoptotic proteins actions. Programmed cell death can be accelerated by Bax

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through binding and antagonizing the BCL-2 or its adenovirus homolog. 10


20, 29

So we then

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examined the influence of samples on Bcl-2 family proteins. As shown in figure 5 E and F,

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vancomycin could dramatically induce the up-regulation of Bax and the downregulation of Bcl-2.

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A significant increase of the bcl-2 level and decrease of the Bax level were observed in the

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DHA-PC group compared to vancomycin group. The levels of Bcl-2 and Bax in DHA group

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exhibited slight alteration when compared to vancomycin group. The levels of Bcl-2 in the PC

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group were not changed in comparison with the vancomycin group. A previous study by Dai et al.

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suggested that cilastatin attenuated vancomycin-induced nephrotoxicity via decreasing Bax/Bcl-2

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ratio. This conclusion was in accordance with our experimental results.

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Effects of different samples on MAPKs signaling pathways

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The MAPKs (ERK, p38, and JNK) play important roles in inflammation, apoptosis, and

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caspase activation in kidney damage induced by vancomycin.29, 38 JNK and p38 could regulate

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various vital cellular functions including cell growth, differentiation, survival and apoptosis.39 The

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blocking of JNK and p38 cascade plays a vital role in mediating the protective effect of kidney

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against oxidative cytotoxicity.

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DHA-PC on protein expressions of JNK and p38 in vancomycin-induced apoptosis. Western blot

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results revealed that vancomycin could significantly induce JNK and p38 phosphorylation

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compared with normal group (P < 0.05) (Fig. 6). These changes were significantly reduced by

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DHA-PC and DHA. However, the levels of p-JNK/JNK and p-P38/P38 after treatment with PC

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alone were not changed in comparison with the control group. Recent reports have indicated that

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cisplatin injection in mice caused a severe nephrotoxicity with phosphorylation of JNK and p38.

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Furthermore, Lu et al. found that methylseleninic acid prevented patulin-induced nephrotoxicity

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via inactivation of MAPKs with decreasing phosphorylation of p38 and JNK, indicating MAPK

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signaling pathways might be related to acute renal failure.29 Our findings provided a mechanistic

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support for DHA-PC protecting against vancomycin-induced nephrotoxicity.

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Effects of different samples on vancomycin-induced activities of antioxidant

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enzymes

40

Therefore, we examined the effects of traditional DHA, PC and

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Oxidative stress is one of the factors that causes mitochondrial dysfunction.41 ROS serves as

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intracellular signaling molecules to control the activation of mitogenactivated protein kinases 11



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(MAPKs), which may play a vital role in drug-induced acute nephrotoxicity.6 Studies have

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revealed that vancomycin administration could result in the over production of free radicals, 42 and

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lead to oxidative damage and lipid peroxidation in renal tissues. Antioxidant enzymes including

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CAT, GST, GSH, and GPx are generally accepted as the second defense line against oxidative

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kidney injury.43 CAT catalyzes the decomposition of hydrogen peroxide, a very vital enzyme in

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protecting the cell from oxidative damage.44 GSH, as a cofactor or a coenzyme, is involved in the

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enzymatic detoxification reaction for oxidative stress.45 GPx, an enzyme family with peroxidase

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activity, is mainly to protect the organism from oxidative damage.46 The biochemical functions of

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glutathione peroxidase mainly include the reduction of lipid hydroperoxides to their corresponding

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alcohols and the reduction of free hydrogen peroxide to water. MDA is usually measured in kidney

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tissue to indicate the lipid peroxidation level. Therefore, we evaluated the effects of samples on

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vancomycin-induced oxidative stress in the kidneys. As shown in Fig 7, vancomycin significantly

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promoted formation of MDA and reduced activities of antioxidant enzymes including CAT, SOD,

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and GPx, and T-AOC in the kidneys. Notably, both DHA-PC and traditional DHA treatment

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significantly suppressed vancomycin-induced renal MDA level and attenuated the depletion of

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antioxidant defense system in comparison with vancomycin group, in which DHA-PC exhibited

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the best effect among three experimental groups. In addition, only GSH activity in the PC alone

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group was slightly increased when compared to the vancomycin group. The levels of CAT, SOD

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and T-AOC after treatment with PC alone were not changed compared with the vancomycin group.

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Previous studies indicated that DHA exhibited a strong protective effect on gentamicin-induced

316

nephrotoxicity and oxidative damage in rat kidney. Furthermore, oral PC pretreatment could

317

protect kidney against cisplatin-induced toxicity and oxidative stress in rats.10 Interestingly, our

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results were not consistent with previous studies. The present research indicated that PC had no

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effect on vancomycin induced acute kidney injury, this might attribute to the short intervention

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time of PC and the differences of dose. Moreover, the drug toxicity of vancomycin was different

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from those of cisplatin and gentamicin.47,

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protected the kidneys against vancomycin through inhibiting oxidative stress.

48

These results indicated that DHA-PC treatment

323

According to the above results, the short-term supplement of DHA-PC had a significant

324

protective effect on acute renal injury induced by vancomycin, which was superior to traditional 12


Page 12 of 30

Page 13 of 30


325

DHA and PC alone. We hypothesized that the outstanding protective effect of DHA-PC on

326

vancomycin induced acute renal injury might be due to its specific molecular structure. DHA-PC

327

includes one molecule of PC at the sn-3 acyl moiety, and one molecule of DHA is located at the

328

sn-2-acyl moiety, which can be released by phospholipase A2. The main forms of DHA are ethyl

329

esters, triglycerides and phospholipids. Increasing evidences showed that phospholipids might be

330

a more effective delivery form of n-3 PUFAs to body tissues than other forms.49, 50 Phospholipids

331

are good emulsifiers that can promote the formation of chyle particles, which is helpful to increase

332

the ability of transporting fatty acids such as DHA.18 Interestingly, it has recently been identified

333

that the deliver of n-3 PUFAs into the body was transported in lyso-PC form rather than

334

un-esterified fatty acids51.

335

In conclusion, DHA-PC is capable of protecting against vancomycin induced kidney injury

336

through mechanisms involved in the inhibition of oxidative stress and apoptosis signaling

337

pathways, as shown in figure 8. Our results indicated that DHA-PC had the potential to be

338

developed into natural adjuvants or functional foods to relieve vancomycin-induced side effects in

339

patients undergoing chemotherapy, and they should be analyzed through further clinical

340

validations and investigations in the future.

341

Funding

342

This work is supported by the National Natural Science Foundation of China [No. 31371757],

343

State Key Program of National Natural Science of China [Grant No. 31330060], National Natural

344

Science

345

Centers[U1606403], and the Fundamental Research Funds for the Central Universities [Grant

346

No.201762028].

347

Notes

348

The authors declare no competing financial interest.

Foundation

of

China-Shandong

Joint

Fund

for

349

13


Marine

Science

Research


350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368

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490

Figure Legends:

491

Table1 Main fatty acid compositions of DHA-PC. Note: DHA-PC (docosahexenoic

492

acid-enriched phosphatidylcholine)

493

Table2 Biochemical parameters in each mouse. Note: N (normal group); M (model group);

494

PC (phosphatidylcholine group); DHA (traditional docosahexenoic acid group); DHA-PC

495

(docosahexenoic acid-enriched phosphatidylcholine group). Values are expressed as mean ±

496

S.E.M * Significantly different compared to controls (P < 0.05). Small letters indicate significant

497

difference at P

Protective Effects of DHA-PC against Vancomycin-Induced

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