Peptides from Antarctic Krill (Euphausia superba) Improve

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Bioactive Constituents, Metabolites, and Functions

Peptides from Antarctic Krill (Euphausia superba) improves osteoarthritis via inhibiting HIF-2# mediated death receptor apoptosis and metabolism regulation in osteoarthritis mice Kai Wang, Yuanyuan Li, Yufeng Dai, Lihua Han, Yujie Zhu, peng wang, and Jingfeng Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05841 • Publication Date (Web): 24 Feb 2019 Downloaded from http://pubs.acs.org on February 26, 2019

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Journal of Agricultural and Food Chemistry

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Peptides from Antarctic Krill (Euphausia superba) improves

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osteoarthritis via inhibiting HIF-2α mediated death receptor

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apoptosis and metabolism regulation in osteoarthritis mice

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Kai Wang, Yuanyuan Li, Yufeng Dai，Lihau Han, Yujie Zhu, Peng Wang*, Jingfeng

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Wang*

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College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province,

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266003, China

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ABSTRACT

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Osteoarthritis (OA) is a prevalent debilitating disease which major characterized

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by cartilage degeneration. In the current study, the destabilization of the medial

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meniscus (DMM) mouse model was used to investigate the effects of Antarctic krill

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Peptides (AKP) on cartilage protection. As observed, AKP clearly ameliorate the

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cartilage degeneration as evidenced by increased cartilage thickness and cartilage area

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and decreased histological Osteoarthritis Research Society International (OARSI)

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scores. Toluidine Blue staining showed that AKO remarkably inhibited the loss of

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cartilage matrix in mice with OA. Hypoxia-inducible factor-2α (HIF-2α) has a key

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role in catabolic regulation and inflammation cascades which are the main causes of

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OA. AKP could down-regulated the expression of HIF-2α and its downstream genes

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such as MMP-13, Adamts-5, IL-1β, iNOS, CXCL-1 and NOS2. Consist with this, the

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anabolic genes such as Acan and Col2α1 were resorted after treatment with AKP.

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Chondrocytes apoptosis and the reduction in cartilage cell viability are also involved

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in the process of OA. HIF-2α mediated death receptor apoptosis signaling pathway

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have been involved in regulation of chondrocyte apoptosis. AKP could reduce the

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expressions of key pro-apoptosis genes in Fas-FasL and DR3-DR3L signaling

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pathways such as Fas, FasL, FADD, Caspase8, caspase3, DR3, DR3L, RIP and

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NF-κB. In addition, expressions of anti-apoptosis genes such as c-AIP and c-FLIP

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were increased significantly. Furthermore, the level of key genes for autophagy,

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including ATG-5, LC-3B and Beclin-1, increased significantly after treatment with

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AKP. These findings indicate that AKP can be used as a new functional factor in the 2



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development of functional foods and chondroprotective drugs.

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KEYWORDS: Osteoarthritis, Peptides from Antarctic Krill, Cartilage metabolism,

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HIF-2α, Death receptor apoptosis

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INTRODUCTION

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Osteoarthritis (OA) is a prevalent degenerative disease characterized by disorder

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of articular cartilage metabolism, leading to joint pain and loss of mobility (1).

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Cartilage homeostasis depends on the integrity of extracellular matrix, which secreted

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by chondrocytes(2). Numerous researches have shown that chondrocytes from OA

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patients exhibit high level of oxidant stress, which is the main cause of activation of

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inflammatory cascades (3, 4). The latter is involved in numerous tissue and cellular

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responses in OA, such as disorder of cartilage metabolism, up-regulation of

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pro-apoptotic factors and increase of chondrocyte loss(5). Apoptosis refers to a

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physiological process aimed to maintain homeostasis of cell, which controlled by

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apoptosis-related genes(6). However, apoptosis of chondrocyte in articular cartilage is

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involved in the OA pathological process and its level positively correlates with the

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severity of cartilage degeneration in OA patient(7, 8). There are two major apoptosis

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regulating mechanisms: mitochondria apoptosis signaling pathway (intrinsic pathway)

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and death ligands apoptosis signaling pathway (extrinsic pathway)(9). During

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pathological process of OA, extrinsic pathway play the major role in chondrocyte

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apoptosis(10). The Fas/FasL signaling pathway is a well-known extrinsic chondrocyte

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apoptosis signal transduction since chondrocytes in OA patient and OA explant 3


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express the Fas receptor, activation of this pathway has been shown to mediate

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apoptosis in osteoarthritic chondrocytes(8). DR3 is another apoptotic receptor

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belonging to the TNFR superfamily, and its expression takes part in the activation of

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apoptosis and proliferation trigger events(11). In addition, Giunta et al. found that the

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endogenous neuropeptides pituitary adenylates cyclase-activating polypeptide

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(PACAP) is a potential chondroprotective agent for OA via the mechanisms by

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inhibiting chondrocyte apoptosis and by decreasing the level of inflammatory factors,

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which indicated that endogenous regulatory targets of chondrocyte play an important

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role in OA pathological process(5).

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HIF-2α (also has been recognized as endothelial PAS domain protein-1) is a key

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heterodimeric transcription factor of the helix-loop-helix/PAS transcription factor

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family(12). Current study showed that HIF-2α is sensitive to oxidative stress,

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inflammatory cytokines, growth factors and unstable biomechanics stress during OA

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pathological process (13). Yang et al. reported that HIF-2α level was enhanced in

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cartilage of OA patient and directly promotes the expression of catabolic genes such

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as MMP-3, MMP-13, adamts-4, Adamts-5, which triggered articular cartilage

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degeneration in DMM mouse model(14). Furthermore, HIF-2α has been regarded as

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the upstream regulator of pro-inflammatory factors in OA pathology(15, 16). Except

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catabolic regulation, HIF-2α has been shown to induce Fas-mediated chondrocyte

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apoptosis and actively potentiated cartilage degeneration during OA pathological

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process. Ryu et al. reported that Fas-knockout mice exhibited less chondrocyte

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apoptosis and milder OA cartilage degeneration after DMM surgery or injection of 4



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Ad-Epas1(17). These studies indicated that HIF-2α may be a potential target for the

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treatment of OA.

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According to the survey of WHO, there are at least 150 million patients with OA

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among the world and the annual costs for OA treatment surpass 190 billion dollars (18,

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19). Current management strategies for cartilage degeneration in OA include

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pharmacotherapy (especially non-steroidal anti-inflammatory drugs, salicylates and

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capsaicin), physical activity and even surgery therapy(20). Unfortunately, oral

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pharmacotherapy have obvious side effects on gastrointestinal track, cardiovascular

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system, liver and renal tissue(21). Due to the anti-inflammatory and lubricating

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properties of physical activity, it has been widely used as a non-pharmacologic treatment

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for OA(22). Musumeci et al. reported that the level of lubricin in rat synovial fluid was

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elevated after physical activity and moderate mechanical joint loading, which ameliorate the

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of lubricative and tribology properties of cartilage, leading to improvement of articular

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cartilage structure and inhibition of cartilage destruction(23). However, it’s hard to restore

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the damaged cartilage and promote cartilage regeneration through physical activity

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program. Nutritional interventions and its application in medical treatment of cartilage

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destruction has currently been the hotpot in OA researches. Wang et al. found that

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functional oil from Antarctic krill can promote cartilage regeneration and matrix

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synthesis as well as significantly inhibit abnormal apoptosis of chondrocytes(24).

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Sampson et al. reported that teriparatide has a high chondro-regenerative activity in

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injury-induced OA(25). Bioactive peptides from marine resource has been shown to

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have antioxidant and anti-inflammatory effects(26). Antarctic krill (Euphausia 5


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superba) is a kind of plankton mainly distributed along Antarctic Ocean and its total

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biomass is estimated to be over 800 million tons(27). Moreover, the protein of

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Antarctic kill contains of whole essential amino acid of mankind and the biological

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value of protein is superior to meat and milk proteins(28). Therefore, the high-value

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utilization of Antarctic krill resource has important significant. Until now, the effect

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of Antarctic Krill peptides on osteoarthritis is still unknown, and the relationship

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between AKP and death receptor apoptosis mediated by HIF-2α has not been reported.

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Therefore, our study may provide a theoretical basis for the use of AKP as a potential

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chondroprotective agent or nutraceutical.

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MATERIALS AND METHODS

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Materials and reagents

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Neutral proteinase FG (Bacillus subtilis 1398) was provided by Enzyme

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Engineering Lab in Ocean University of China (Qingdao, China). Eva-Green master

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mix, TRIzol and protein prestained marker were purchased from Thermo Scientific

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(Thermo Fisher Scientific Ltd, MA, USA). EasyScript Plus cDNA Synthesis Kit was

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product of ABM Co. Ltd (Richmond, BC, Canada). Rabbit anti-mouse HIF-2α, IL-1β,

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FasL, caspase3, cleaved-caspase3, NF-κB p65, DR3L, phosphorylated NF-κB p65

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(p-NF-κB p65) and β-actin were from Abcam (Cambridge, UK). The primers of all

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genes (Table S2) were provided by Genewiz Biotech Co. Ltd (Suzhou, China).

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Preparation of AKP

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AKP was provided by Dr. Yanchao Wang from Food and Human health 6



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laboratory in Ocean University of China (Qingdao, China). Briefly, powder of

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Antarctic krill was defatted with 90% ethanol at 25 °C, then the ethanol was removed

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through vacuum drying at 65 °C for 2 h. Neutral proteinase FG (Bacillus subtilis 1398)

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was added in a ratio of 2% (E/S) and hydrolyzed at 45°C for 5 h. The AKP was

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obtained by spray drying after de-fluorination for 24 h. The protein content of AKP

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was 82.5% and the fluorine content of AKP was 5.36 mg/kg. The molecular weight

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distribution of peptides in AKP was from 200 to 2000 Da.

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Determination of amino acid composition

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Amino acid composition was analyzed according to method of Cohen and

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Strydom(29) after acidic hydrolysis in 6 M HCl and pre-derivatization with

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phenyl-isothiocyanate. Tryptophan was determined after basic hydrolysis with

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Na(OH)2 as previously described(30). Total cysteine content was detected by the

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Institute of Oceanology, Chinese Academy of Science (Qingdao, China).

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Animals and osteoarthritis model

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This animal experiment was approved by the Ethical Committee of Experimental

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Animal Care of the Ocean University of China (certificate no. SYXK20120014).

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C57BL/6 male mice (8 weeks old, 20±1g) were purchased from the Vital River

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Laboratory Animal Center (Beijing, China). Mice housed in pathogen-free

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environment with 5 animals each cage. The animal room was under a 12 h light/dark

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cycle with a constant temperature from 22 °C to 24 °C. Mice are free to get food and

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water.

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After acclimatization for 3 days, DMM surgery was performed as previously 7


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described(31). Briefly, the joint capsule of experimental mice was opened after

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anesthesia with 1% pentobarbital sodium, then fat pad was dissected bluntly to expose

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the menisco-tibial ligament in media side (MMTL), which was then transected by

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microsurgical knife (11#). Joint capsule of sham control mice was opened but the

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MMTL was left intact. The joint capsule and skin were closed with absorbable sutures.

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All mice were free to move in their cages after DMM surgery. After 8 weeks of

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surgery, the mice were divided into Sham control group (physiological saline), DMM

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control group (physiological saline), GLU positive group (glucosamine sulfate, 195

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mg/kg·Body Weight (BW)), AKP-L group (AKP, 400 mg/kg·BW) and AKP-H group

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(AKP, 600 mg/kg·BW) of 30 animals each. A volume of 10 mL/kg·BW was

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intragastrically administered to mice of all groups once a day for 8 weeks. Mice were

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euthanized after eight weeks of intragastrical administration by the American

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Veterinary Medical Association approved way(32) and the right entire joints were

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harvested for histological analysis, qRT-PCR analysis and Western Blot

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determination.

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Histological analysis and osteoarthritis score

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The right knee joint was fixed for 24 h with 4% paraformaldehyde, then

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embedded into paraffin after decalcification with 10% EDTA. Sagittal sections

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(5-μm thickness) were stained with Hematoxylin & Eosin (Solarbio Biotech Co. Ltd,

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Beijing, China) and Toluidine Blue (Sigma-Aldrich, Poole, UK), respectively. The

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cartilage histological observation was performed on BH-2 microscope system

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(Olympus, Tokyo, Japan). The cartilage thickness in weight-loading area, including 8



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hyaline cartilage (HC) and calcified cartilage (CC) were determined by ImageJ

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software (National Institutes of Health). The part of the cartilage surface to the

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tidemark is defined as hyaline cartilage (HC). The part of the tidemark to

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cartilage/bone junctions is defined as hyaline cartilage. Total cartilage thickness is

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the sum of the two parts. Total area of articular cartilage was calculated by the

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OsteoMetrics system via toluidine blue-stained sections, as previously described(25).

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The degree of cartilage erosion was evaluated using the osteoarthritis research

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society

international

(OARSI)

semi-quantitative

scoring

based

on

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histomorphology(33). As shown in Table S1, scoring protocol was performed in

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blinded fashion by three independent specialists.

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Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

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analysis

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Frozen cartilage (50mg) was homogenized with liquid nitrogen, then extraction

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and purification of RNA was performed as previously described using TRIzol

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regent(34). The content and quality of RNA was determined by NanoDrop 2000

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spectrophotometer (Thermo-fisher Scientific, MA, USA). Subsequently, the

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single-stranded cDNA was reversed from RNA according to the protocol of

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EasyScript Plus cDNA synthesis Kit. Then, qRT-PCR analysis was performed on the

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LightCycler 96 system (Roche Inc., Hercules, CA, USA) with EvaGreen Master Mix.

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The thermal conditions are as follows: initial denaturation at 95°C for 10 min, 45

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cycles of denaturation at 95°C for 15 s, annealing at 60°C for 10 s, and extension at

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72°C for 45 s. All the mRNA levels were normalized to β-actin and each qRT-PCR 9


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reaction was determined in triplicate. Relative quantification of genes level was

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performed using standard curve method.

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Wsetern Blot

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The protein levels of HIF-2α (61 kDa), FasL (31 kDa), caspase3 (32kDa),

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cleaved-caspase3 (17 kDa), DR3L (44 kDa), NF-κB p65 (65 kDa), p-NF-κB p65 (65

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kDa), IL-1β (17 kDa) were examined by western blotting. Frozen cartilage (80mg)

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was homogenized with liquid nitrogen and total protein was obtained using Western

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and IP lysis buffer (Applygen, Beijing, China). Then 10-12% SDS-PAGE was used to

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separate the target protein according to different molecular, followed electrically

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transferred onto 0.45 polyvinylidene ﬂuoride (PVDF) membranes (GE Healthcare,

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Wisconsin, USA). After blocking with 5% Bovine Serum Albumin (BSA), the

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membranes were incubated overnight at 4 °C with primary antibodies against

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FasL(1:1000), HIF-2α(1:1000), caspase3(1:1500), cleaved-caspase3(1:1500), NF-κB

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p65(1:2000), p-NF-κB p65(1:2000), DR3L(1:800), IL-1β(1:1000), and β-actin

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(1:2000). After incubation with horseradish peroxidase (HRP) conjugated secondary

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antibodies for 2 h, the protein bands were visualized using a superECL stain kit

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(Applygen, China) and the gray intensity was calculated by Image J program

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(National Institutes of Health). The protein level of β-actin was used as internal

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control.

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Statistical analysis

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Data were presented as mean ± standard error of mean (SEM). Statistical

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analysis of means among different groups was performed using one-way analyses of 10



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variance (ANOVA) followed by Duncan’s multiple comparisons using the SPSS

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software. P < 0.05 were considered statistically significant.

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RESULT

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Amino acid composition of AKP

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The amino acid composition of peptides play a significant role in various

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physiological process(35). As shown in Table 1, Glutamic acid (14.8%) and aspartic

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acid (11.1%) were predominant amino acid of AKP. Hydrophobic amino acids,

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including GLY, ALA, VAL, PRO, TRP, ILE, MET, LEU and PHE, accounted for a

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proportion of 43.13%. In addition, AKP also comprised a high content of essential

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amino-acid (THR, ILE, LEU, VAL, LYS, TRP, PHE, MET), which accounted for

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40.98%.

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AKP ameliorated the cartilage structure in DMM model mice

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Hematoxylin & Eosin and Toluidine Blue staining showed that sever cartilage

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degeneration and loss of cartilage proteoglycan occurred in the DMM control group

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compared with the sham control group (Fig.1A). Compared to the DMM control

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group, there was less proteoglycan loss and milder cartilage erosion in AKP-L and

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AKP-H group. As shown in Fig. 1B, quantitation of total cartilage thickness and area

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in the AKP-H group were increased by 43.70%, and 76.69%, respectively (P < 0.01).

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The ratio of HC/CC was enhanced by 189.25% in the AKP-H group (P < 0.01).

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Correspondingly, semi-quantitative OARSI scores in the AKP-H group were

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significantly lower than that in the DMM control group (P < 0.01). These data suggest

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a chondroprotective effect of AKP on osteoarthritis mice and inhibition of cartilage 11


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matrix loss in DMM-induced OA.

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AKP decreased the level of HIF-2α and regulated downstream catabolic genes in

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articular cartilage

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HIF-2α is controlled via oxygen-dependent degradation and is involved in

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regulation of oxidative stress and cartilage homeostasis(13). As shown in Fig. 2, the

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mRNA and protein level of HIF-2α was remarkably elevated in the DMM control

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group. Correspondingly, the expression of downstream catabolism genes such as

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MMP-13 and Adamts-5 were increased significantly in the DMM control group (P

Peptides from Antarctic Krill (Euphausia superba) Improve

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