Arginyl-fructosyl-glucose, a Major Maillard Reaction Product of Red

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Bioactive Constituents, Metabolites, and Functions

Arginyl-fructosyl-glucose (AFG), A Major Maillard Reaction Product of Red Ginseng, Attenuates Cisplatin-Induced Acute Kidney Injury by Regulating NF-#B and PI3K/Akt Signaling Pathways Rong-yan Li, Wei-zhe Zhang, Xiaotong Yan, Jingang Hou, zi wang, Chuanbo Ding, Wen-cong Liu, Yinan Zheng, Chen Chen, Yue-ru Li, and Wei Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00540 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 4, 2019

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Arginyl-fructosyl-glucose (AFG), A Major Maillard Reaction Product of Red

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Ginseng, Attenuates Cisplatin-Induced Acute Kidney Injury by Regulating

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NF-κB and PI3K/Akt Signaling Pathways

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Rong-yan Li1, Wei-zhe Zhang1, Xiao-tong Yan1, Jin-gang Hou1,2, Zi Wang1, 3, Chuan-bo Ding1, Wen-

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cong Liu1, 3, Yi-nan Zheng1, Chen Chen4, Yue-ru Li1, and Wei Li1, 3*

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1

College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China

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2.

Intelligent Synthetic Biology Center, Daejeon 34141, Republic of Korea

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3.

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Changchun 130118, China

National & Local Joint Engineering Research Center for Ginseng Breeding and Development,

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4

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Abbreviations：

School of Biomedical Sciences, University of Queensland, Brisbane 4072, Australia

CDDP

Cisplatin

AKI

Acute kidney injury

AFG

Arginyl-fructosyl-glucose

CRE

Creatinine

BUN

Blood urea nitrogen

H&E

Hematoxylin and Eosin

MDA

Malondialdehyde

GSH

Glutathione

CAT

Catalase

SOD

Superoxide Dismutase

CYP2E1

Cytochrome P450 E1

4-HNE

4-hydroxynonenal

iNOS

Inducible nitric oxide synthase

TNF-α

Tumor necrosis factor-α

IL-1β

Interleukin-1β

IL-6

Interleukin-6

PI3K

Phosphatidylinositol 3-kinase

Akt

Protein kinase B

IκB

Inhibitor of κB

Nuclear factor kappa B

Iκκ

IκB kinase

Bcl-2

B-cell-lymphoma-2

Bax

B-associated X

qPCR

Quantitative PCR

NF-κB

COX-2

LC-MS/MS

Cyclooxygenase-2

Liquid chromatograph-mass

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spectrometer Running title: AFG ameliorates CDDP-induced nephrotoxicity

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Correspondence

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Professor Wei Li, College of Chinese Medicinal Materials, Jilin Agricultural University,

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Changchun 130118, China. E-mail: [email protected] (W. Li) Tel. /Fax: +86-431-84533304.


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ABSTRACT:

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Recently, although ginseng (Panax ginseng C. A Meyer) and its main component saponins

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(ginsenosides) have been reported to exert protective effects on cisplatin (CDDP)-induced acute

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kidney injury (AKI), the beneficial activities of non-saponin on CDDP-induced AKI is little

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known. This research was designed to explore the protective effect and underlying mechanism

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of Arginyl-fructosyl-glucose (AFG), a major and representative non-saponin components

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generated during the process of red ginseng, on CDDP-caused AKI. AFG at doses of 40 and

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80 mg/kg remarkably reversed CDDP-induced renal dysfunction, accompanied with the

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decreased levels of serum CRE and BUN. Interestingly, all of oxidative stress indices were

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ameliorated after pretreatment with AFG continuously for ten days. Importantly, AFG relieved

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the CDDP-induced inflammation and apoptosis in part by mitigating the cascade initiation steps

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of NF-κB signals and by regulating the participation of PI3K/Akt signal pathway. In conclusion,

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these results clearly provide a strong rationale for the development of AFG to prevent CDDP-

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induced AKI.

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Key words: Arginyl-fructosyl-glucose, cisplatin, acute kidney injury, PI3K/Akt, NF-κB.

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1. Introduction

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Cis-diamminedichloroplatinum (cisplatin, CDDP) , one of the classic chemotherapeutic agents,

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which is widely applied for patients for killing various malignant tumors, including cervical

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cancer, ovarian cancer, lung cancer, neck cancer, and other severe cancers(1). However,

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increasing evidence proves severe side effects of CDDP, including nephrotoxicity electrolyte

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imbalance, myelotoxicity and ototoxicity, as main challenges in CDDP-based cancer therapy(2).

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In clinical CDDP-treated patients, acute kidney injury (AKI) has become the most common and

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serious complication. Pathophysiologically, the accumulation of CDDP in straight proximal

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and distal curved tubules in kidney, leading to a rapid decline in renal functions(3).

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Indeed, several studies have revealed that CDDP-induced AKI is a very complex multifac-

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torial pathophysiological process(4). Increased oxidative stress caused by imbalance between

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oxidation and anti-oxidation in vivo plays important roles in the CDDP-induced kidney injury,

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through increase in reactive oxygen species (ROS) production and resultant renal dysfunction,

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inflammation response and apoptosis(5). It is well known that oxidative stress stimulates tran-

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scription factors such as nuclear factor-kappa B (NF-κB)(6). As a consequence, the activation

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of NF-κB results in the increased expression of a serious of pro-inflammatory cytokines, such

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as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase

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(iNOS), and cyclooxygenase-2 (COX-2)(7). Although the precise mechanism of CDDP-induced

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AKI remains unclear, apoptosis has been considered as a main cause of nephrotoxicity(8).

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PI3K/Akt pathway is critically important in regulating the cell functions, including growth,

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proliferation, and survival(9). Actually, there have been reports showing modifications of

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PI3K/Akt signaling pathway in CDDP-inducted AKI(10). Once PI3K/Akt signaling pathway is

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aberrantly activated, it will cause oxidative stress, apoptosis and inflammatory response, con-

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sisting CDDP pathophysiology. Accordingly, available data revealed that regulating oxidative

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stress, inflammatory and apoptosis played a key role against CDDP-induced AKI(11).

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Accumulated research results have proven that natural compounds of medical herbs are able

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to treat CDDP-induced AKI. Red ginseng (Panax ginseng Meyer), produced from fresh ginseng

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through the processes of steaming and drying via Maillard reaction (MR), possesses multiple

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biological effects which include inhibit tumor growth, decrease blood glucose levels,

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antioxidant activity and others(12, 13). Furthermore, red ginseng is enriched with amino acids and

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sugars. It is clear that red ginseng has a wide variety of pharmacological effects mainly caused

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by ginsenosides(14, 15). However, less attention has been paid on non-saponins of red ginseng

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extracts. Arginyl-fructosyl-glucose (AFG, Fig. 1A) is one of the representative non-saponins

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in red ginseng, which accounts for 3~4% of red ginseng (dry weight basis)(16). It was reported


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that AFG exerted good antioxidant properties(17), and inhibition of synthesis of pro-

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inflammatory cytokines both in vitro and in vivo(18, 19). Moreover, administration of AFG caused

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anti-hyperglycemic effect(20). Although red ginseng and saponins as active constituents have

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strong protective effects on kidney injury(21), it is important to explore whether AFG as a major

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non-saponin contributes to the protective effects of red ginseng.

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Based on accumulated evidence that AFG exerted better antioxidant and anti-inflammatory

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effects in kidney, the present research was performed to demonstrate that AFG was capable of

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preventing CDDP-inducted AKI. Regulation of PI3K/AKT and NF-κB signaling pathways

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maybe the potential molecular mechanism of AFG action.

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2. Material and methods

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2.1 Chemical compounds and Reagents

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CDDP was acquired from Sigma-Aldrich (St. Louis, MO, USA). Hematoxylin and eosin

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(H&E) and plasma and tissue biochemical assays kits for measuring creatinine (CRE), blood

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urea nitrogen (BUN), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde

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(MDA), catalase (CAT) were purchased from Nanjing Jiancheng Bioengineering Institute

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(Nanjing, China). Terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL)

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apoptosis detection kits were provided by Beyotime Biotechnology (Shanghai, China). SABC-

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DyLight488-labeled and secondary antibodies were provided by BOSTER Biological

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Technology (Wuhan, China). UNIQ-10 Column Ttizol Total RNA extraction Kit and Tap PCR

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master Mix (2×, blue dye) were bought from Sangon Biotech (Shanghai, China); Special

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reagents for quantitative PCR (qPCR) kits SYBR® Premix Ex TaqTM II were acquired from

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TaKaRa Bioengineering Institute (Dalian, China). The antibody of rabbit monoclonal anti-

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mouse 4-hydroxynonenal (4-HNE), Cytochrome P450 E1 (CYP2E1), inducible nitric oxide

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synthase (iNOS), cyclooxygenase-2 (COX-2), and HRP-conjugated anti-mouse IgG were all

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obtained from Abcam (Cambridge, UK). phosphatidylinositol 3-kinase (PI3K,p85), Protein

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kinase B (Akt), p-PI3K (p-p85), p-Akt, b-associated X (Bax), (b-cell-lymphoma-2) Bcl-2,

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caspase-3, cleaved-caspase-3, IκB kinase α/β (IKKα/β), inhibitor of κBα (IκBα), nuclear factor-

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kappa B (NF-κB,p65), p-IKKα, p-IKKβ, phospho-IκBα (p-IκBα), phospho-NF-κB (p-NF-κB,

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p-p65), COX-2, iNOS, β-actin and secondary antibodies for western blot were all received from

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Cell Signaling Technology (Danvers, MA, USA).

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An reference standard of AFG was isolated and purified from red ginseng (Panax ginseng

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C.A Meyer), with purity of 98.5%, as previously reported using high-performance anion-

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exchange chromatography through integrated pulsed ampere metric detection (HPAEC-

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PAD)(22). All other chemicals were bought from Beijing Chemical Works (Beijing, China).



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2.2 Preparation of AFG

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AFG was prepared by modified method developed by Prof. Yi-Nan Zheng et al.(23) The

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method is reacted with arginine and maltose under anhydrous acidic conditions at 80 °C for 120

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min.

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We used the process of purifying the Maillard reaction product (MRP)-AFG in combining

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cation exchange resin with polyacrylamide gel column chromatographic method. First, the

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crude product of AFG was appended to the cation exchange resin column with 1/5 times of

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resin volume and eluted by the distilled water to a neutral stance. Neutralized with distilled

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water, it was then washed with 4% ammonia water using TLC for tracking the AFG. The

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component was collected with value of Rf 0.156 and the ammonium hydroxide removed. A

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portion of the ammonia water was removed by a rotary evaporator, then, the lyophilized powder

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was diluted with sterile water to a 0.5 g·mL-1 super polyacrylamide gel column (Bio-gel P-II).

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The AFG single point was collected and freeze-dried.

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2.3 Determination of AFG by UPLC-MS/MS

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The present method was performed as indicated in literature with minor modifications.

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UPLC were consisted of a Shimadzu LC-30AD system (Tokyo, Japan) and Sciex 4500 system

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(Applied Biosystems Inc., Foster City, CA). AFG was separated on the Waters Acquity UPLC

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BEH Amide AAA column (100 mm ×2.1 mm, 1.7 μm) at 40 °C. The mobile phase was coupled

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with a gradient elution system of solvent A (0.01 mol/L ammoniumformate and methanol buffer

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solution, pH=6.5) and solvent B (Acetonitrile:H2O=4:1, v/v). The flow rate was set at 0.3

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mL/min (0.0 min, 0% B phase; 2 min, 2% B; 10 min, 5% B). The MS/MS detection was used

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positive electrospray ion fragmentation pattern. Multiple reaction monitoring (MRM) mode

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was performed to the precursor to production transitions, with a dwell time of 150 ms. The

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optimum conditions were set as following: curtain gas (N2): 35 psi; ion spray voltage 5500 V;

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heated nebulizer temperature 550 °C; nebulizing gas (N2) and heater gas (N2) 55 psi. It was

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finally determined to be AFG. Data were acquired with Peak View® Software (Applied

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Biosystems Inc., Foster City, CA).

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2.4 Animals and Drug treatment

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ICR mice, male, 8 weeks old, weighting 20-22g, were used for the study, which obtained from

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YISI Experimental Animals Co. Ltd with Certificate No.: SCXK (JI) 2016-0003 (Changchun,

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China). All animals were acclimatized for one week at a standard rodent diet, humidity (60 ±

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10 %), and temperature (25.0 ± 2.0 °C), with 12/12 h light dark cycle. The experimental

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protocol was executed strictly approved by the Guide for the Care and Use of Laboratory, which

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was approved by the Ethical Committee for Laboratory Animals of Jilin Agricultural University.


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For this experiment, all animals were divided into four groups in a random manner: control

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group, CDDP group (20 mg/kg) and two AFG groups (40 and 80 mg/kg). After adaptive feeding,

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AFG was dissolved with 0.9% physiological saline and administered intragastrically to mice

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for 10 consecutive days. The other two groups were given 0.9% saline. On the 7th days, mice

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were injected CDDP (20 mg/kg, diluted 0.9 % warm saline) by intraperitoneal for one time to

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all the groups except for normal group 1h after the final AFG treatment. The experiment was

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terminated 72 h after injection of CDDP. The blood samples were collected from eyeball. Then,

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mice were sacrificed by cervical dislocation under anesthesia and dissected rapidly. The serum

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samples were separated by centrifugation (3500 rpm, 10 min), and collected for subsequent

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measurement. Kidneys were collected for the following research.

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2.5 Estimation of CRE and BUN in serum

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Immediately after sacrifice, serum BUN and CRE were evaluated as manufacturer’s protocol

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of the biochemical assay kits (Jiancheng, Nanjing, China).

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2.6 Oxidative stress markers in kidney

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The kidney homogenate was used to estimate the antioxidant activities. Lipid peroxides were

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measured by MDA kits, SOD enzymatic activity, CAT activity and GSH content were

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determined by related assay kits(24).All of these were determined to the manufacturer’s

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protocols(Jiancheng, Nanjing, China). Bradford protein assay kits were used to measure protein

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concentrations (Beyotime Biotechnology, Shanghai, China), using bovine serum albumin (BSA)

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as the standard.

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2.7 Histopathological examinations

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The kidney tissues were kept in 10 % formaldehyde before embedded in paraffin and cut

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into 5μm-thickness sections. Later, slices were rehydrated in decreasing concentrations of

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ethanol and stained with H&E for detecting the changes in histopathological analysis following

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established protocol(25). Tubular damage was evaluated by scoring tubular necrosis in 4

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different fields in the corticomedullary junction (microscopy, Leica DM750, Germany). The

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results were expressed as necrosis score as previously reported(26).

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2.8 TUNEL staining

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Apoptosis was determined by TUNEL assay, which was carried out as previously described

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with slight revision. The In Situ Cell Death Detection Kits (Beyotime Biotechnology) were used

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to measure apoptotic cells. The apoptosis cells were calculated by microscope (microscopy,

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Leica DM750, Germany).

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2.9 Immunohistochemistry and Immunofluorescence staining



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According to the previously described method with minor modification(27), the paraffin slices

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were deparaffinized and rehydrated with xylene and different concentration ethanol, and then

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the sections were permeabilized for anti-genretrieval. Subsequently, the slides were incubated

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for 1.5 h under 1% BSA, and further incubated with primary antibodies to iNOS (1:200) or

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COX-2 (1:200) at 4◦C for 12h. Sections were then through the incubation with horseradish

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peroxidase (HRP) conjugated secondary antibody (Abcam, Cambridge, UK). Then, sections

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were rinsed with distilled water and then slides were analyzed with dispute adjudication board

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(DAB) and counterstained with hematoxylin. The brownish-yellow color in the cytoplasm of

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the renal cells were recognized as positive staining and observed (Leica DM750, Germany),

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and the expression intensity was measured through Image Pro-Plus 6.0 software.

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The sections were incubated with 4-HNE (1:200) or CYP2E1 (1:200) at 4 °C for 10 h in

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immunofluorescence staining, subsequently, hatched by DyLight-488 after 12h at 37°C

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(Abcam, Cambridge, UK). 4, 6 diamidino-2-phenylindole (DAPI) was used to stain the nuclei

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of fixed tissue cells. Microscope (Leica DMILED, Germany) was used to measure

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immunofluorescence staining.

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2.10 Quantitative PCR

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The expression of the genes of interest was determined from kidney by qPCR. RNA was

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isolated with the UNIQ-10 column-based Trizol Total RNA Extraction Kit (Sangon, Shanghai,

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China). Then, total RNA was purified and reversed to cDNA using the Prime ScriptTM RT

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reagent kit (TaKaRa, Dalian, China). qPCR was performed bestowing a Step OneTM Real-Time

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PCR System (Applied Biosystems, Foster City, CA, USA) and amplificatory reactions were

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achieved according to the manufacturer's protocol using SYBR® Premix Ex TaqTM

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(TaKaRa, Dalian, China). The program for amplification was used at 95 °C for 5 min followed

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by 40 cycles of 3-step PCR. The specific primmer sequences acquired from TaKaRa

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Biotechnology (Dalian, China) which are shown in Table 2. The relative expression of β-actin

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was used as an internal control. All mRNA was calculated by 2-ΔΔc (t) method and given as ratio

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compared with the normal group.

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2.11Western blot analysis

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kit

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Western blot was processed with a standard procedure. Proteins from kidneys were extracted

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in RIPA buffer. And then determining the protein concentration according to the methods

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introduced above. Subsequently, and then determining the protein concentration the protein

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samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF)

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membranes. After blocking with BSA for 1.5 h, membranes were incubated at 4°C for 12 h

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with different primary antibodies. The membranes were then incubated with HRP conjugate


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secondary antibodies. The protein bands were detected by the emitter coupled logic (ECL) plus

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western blot detection system with β-actin as the internal reference. Signal intensities were

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measured with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

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2.12 Statistical analyses

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All data were analyzed by using a software of GraphPad Prism 7.0 (ISI, USA), which was

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established on different experiments as mean ± standard deviation (Mean ± S.D.). One-way

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analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical

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significance. P-values less than 0.05 or 0.01 were considered statistically significant.

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3. Results

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3.1 UPLC-MS/MS assay

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According to the previous research, we obtained the crude AFG product under the anhydrous

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environment of arginine and maltose. The synthesis rate reached 80%, therefore it was further

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purified for this study.

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Mass spectrometry was carried out in MRM mode for detection of AFG in red ginseng and

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chemically synthesized. The decluttering potential (DP, 80V), collision energy (CE) and cell

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exit potential (CXP, 11V) were optimized in order to improve the optimum sensitivity and

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selectivity. After optimization on the LC-MS/MS system and based on previous work(28),the

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expected molecular mass of [M+H]+ 499.2 was observed and two MRM ion transitions were

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determined by AFG (Fig.1B). The optimized two fragment ions and CE values for each

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transition were 499.2 → 70.0 (30 V, 93 V) and 499.2 → 112.1 (30V, 35 V) (Fig. 1C). As

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expected, the AFG in red ginseng is in the same fraction with AFG reference, which was

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chemically synthesized. These values matched the theoretical molecular weights previously

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reported. Moreover, AFG was confirmed with a purity of 97.3%, sufficient for the following

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experiments.

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3.2 AFG attenuated CDDP-induced renal dysfunction and histopathological damage

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Kidney tissues from CDDP treatment mice was obviously whitened swollen and

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accompanied by hydropic degeneration, which was remarkably improved by the administration

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of AFG for ten days (Fig. 2A). H&E staining clearly showed that renal tissues in control group

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had clear tubular and glomerular structures with normal nucleus. Serious renal injury was

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manifested by tubular necrosis and glomerular congestion (p < 0.01) after a single CDDP

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exposure. In contrast, pretreatment with AFG for consecutive 10 days dose-dependently

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attenuated pathological changes (p < 0.05 or p < 0.01), leading to lower histopathological score

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than that in CDDP-treated mice (Fig. 2B and C, p < 0.05 or p < 0.01).



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CDDP administration significantly elevate the CRE and BUN's level in serum, indicating a

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serious renal injury (p < 0.01). Nevertheless, after supplement of AFG pretreatment, the

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obviously reduction of the levels of CRE and BUN happened in comparison to that in CDDP-

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treated mice (Fig. 2D and E, p < 0.05 or p < 0.01), indicating that AFG supplementation

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significantly attenuated the CDDP-induced renal injury (p < 0.05, p < 0.01). The results

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indicated that AFG may prevent CDDP-evoked acute kidney injury.

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3.3 AFG attenuated CDDP-induced oxidative stress in kidney

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To assess the functions of AFG against CDDP-induced oxidative stress, the renal levels of

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MDA, SOD, GSH and CAT were assayed with the supernatant of kidney homogenate. Relative

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to control group, the GSH contents (p < 0.05), SOD (p < 0.01) and CAT activity (p < 0.05)

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were clearly reduced by CDDP administration, while the content of MDA was elevated (p

Arginyl-fructosyl-glucose, a Major Maillard Reaction Product of Red

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