Immunomodulatory Effects of Shark Cartilage: Stimulatory or Anti-Inflammatory

Journal Pre-proof Immunomodulatory Effects of Shark Cartilage: Stimulatory or Anti-Inflammatory Elahe Safari

PII:

S1359-5113(19)30934-1

DOI:

https://doi.org/10.1016/j.procbio.2020.01.032

Reference:

PRBI 11917

To appear in:

Process Biochemistry

Received Date:

19 June 2019

Revised Date:

10 January 2020

Accepted Date:

29 January 2020

Please cite this article as: Safari E, Immunomodulatory Effects of Shark Cartilage: Stimulatory or Anti-Inflammatory, Process Biochemistry (2020), doi: https://doi.org/10.1016/j.procbio.2020.01.032

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Immunomodulatory Effects of Shark Cartilage: Stimulatory or Anti-Inflammatory

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Elahe Safari

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Iran University of Medical Sciences, Tehran, 1449614535 Iran

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Highlights:

Shark cartilage is a natural supplement for different diseases Shark cartilage have Anti-tumor and anti-angiogenesis function Shark cartilage have immunomodulation function Protein fraction of shark cartilage stimulate immune system Shark cartilage polysaccharides have anti-inflammatory effects

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Abstract

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immunostimulatory, and anti-inflammatory characteristics, this natural supplement is directly

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or indirectly marketed as a therapeutic and preventive option for various diseases e.g. cancer

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and rheumatoid arthritis (RA). In this study, the immunomodulatory effects of SC were

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reviewed in terms of SC components. Therefore, the effects of total SC without any

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fractionation were explained and low-molecular-weight protein (LMWP) fraction along with

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type II collagen (CII) having stimulatory impacts on the immune system were illustrated.

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Eventually, the immunomodulatory effects of SC proteoglycan (PG) revealed that the given

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fractions were useful for inflammatory diseases.

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Regarding the biological effects of shark cartilage (SC) as well as its anti-angiogenic,

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Keywords: Shark Cartilage, Immunomodulation, Leukocyte, Cytokine, Inflammation

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1. Introduction

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Natural dietary supplements are one form of complementary and alternative medicine used to

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treat various diseases or improve general health. However, no study has so far fully described

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the biological activity of numerous commercially available supplements [1, 2]. Commercial

32

products such as cartilage, chondroitin sulfate, and glucosamine of diverse sources are being

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increasingly accepted as therapeutic and preventative options in this domain. In this respect,

34

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angiogenic qualities and efficiencies as an anti-cancer drug [3, 9] have been examined thus

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far by in vivo studies but in vitro investigations [10, 11] have been merely conducted on its

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biological characteristics or pharmacological ones. Moreover, some evidence has confirmed

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the stimulatory effects of SC on specific components of the immune system [12, 15]. On the

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other hand, other bioactivities of SC such as impacts on osteoarthritis (OA), progressive

40

systemic sclerosis (SSs), rheumatoid arthritis (RA), neurovascular glaucoma (NVG), and so

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forth have been delineated in some studies [16-18]; and the related literature has further

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reflected on anti-inflammatory and wound-healing factors in SC [19]. Therefore, this study

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was conducted to review the biological activity of SC components on immunomodulation to

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understand the efficacy of this supplement.

2. Anti-Tumor and Anti-Angiogenic Effects of SC

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one of the products used as a dietary supplement is shark cartilage (SC) whose anti-

45 46 47 48

formation or angiogenesis are therefore needed for tumors exceeding 1-2 mm in diameter to

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boost their growth [3]. There are no blood vessels in cartilage, i.e. it is avascular; so it rarely

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leads to development of malignant tumors. Cartilage is additionally the first normal tissue

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considered as a natural resource of anti-angiogenic compounds [3]. Moreover, sources of

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several components with anti-angiogenic and anti-tumor activities can be found in cartilage

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from calves (i.e. bovine cartilage) and sharks [3, 4, 20, 22]. Thus, identifying cartilage

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components inducing these anti-angiogenic and anti-tumor effects has drawn scholars

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towards using cartilage-derived molecules in therapeutic applications for tumors.

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The inhibitory effect of shark serum on Lewis lung carcinoma was first stated by Snodgrass

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et al. in the 1970s [23]. It was reported that there were agents in shark serum inhibiting tumor

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growth sharks were viewed as a source of anti-angiogenic agents. As well, cartilage-derived

59

inhibitor (CDI) was introduced in the study by Moses et al. [20]. Anti-angiogenic activity of

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SC extract (SCE) was correspondingly settled by McGuire et al. [24]. Furthermore, the potent

61

anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay was

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demonstrated in a commercially available preparation from blue SC dubbed as U-995.

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Findings had correspondingly indicated that sarcoma growth in mice had not been modified

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by oral administration although intra-peritoneal delivery had been conducted in a dose-

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dependent manner [25]. A significant decrease of lung metastases of B16F10 melanoma had

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Angiogenesis develops newly-formed vessels from former vasculature. New capillary

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prevent metastases.

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It had been found that neovascularization could be inhibited by the liquid SC product AE-941

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(i.e. Neovastat) [22]. As a standardized water-soluble extract, AE-941 had been tested in

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phase III trials in Europe and North America for metastatic non-small-cell lung carcinoma

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(NSCLC) [26]. Tumor volume, number of lung nodules [27], and metastases in Lewis lung

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carcinoma model had been further decreased via administrating Neovastat orally [28].

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Likewise, tumor volume shrinkage had been observed by administrating Neovastat orally in a

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mice model of breast cancer [29]. In addition, patients suffering from unresectable stage IIIA,

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IIIB, or IV NSCLC and receiving Neovastat, had experienced a significant survival

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been similarly reported in intra-peritoneal delivery of U-995, but higher oral dose could also

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angiogenesis inhibition had been reported in many in vitro and in vivo studies. Inhibition of

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basic fibroblast growth factor (βFGF)-induced angiogenesis [28], prevention of collagenase-

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induced collagenolysis [25], increased T cell infiltration into tumor [13, 31], and cell

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adhesion inhibition by modifying focal adhesion proteins had been accordingly proposed as

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mechanisms contributing to anti-angiogenic effects of SC [32]. Furthermore; different

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mechanisms such as matrix metalloproteinase (MMP) inhibition [33], blockage of vascular

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endothelial growth factor (VEGF) binding to its receptor, endothelial cell (EC) apoptosis

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induction, and angiostatin production stimulation [25, 28, 34, 35] through AE-941 had been

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advantage [30]. Finally, the critical role of SC in preventing cancer expansion and

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should be noted that clinical trials of SC against advanced cancer had been triggered by

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positive in vitro and in vivo results, but satisfactory outcomes had not been observed [37]. As

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a single agent, SC was dormant in patients with advanced- stage cancer, so that their quality

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of life had not been improved. In this way, the effectiveness of SC product in patients with

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last stages of colorectal and breast cancers had not been observed in a phase III trial [38].

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As a result, lack of studies approving SC bioavailability when used orally as well as advanced

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stage of cancer in patients passed through clinical trials caused a mismatch between clinical

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trials and in vitro or in vivo studies. Moreover, the failure of Neovastat had been justified as;

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that is, this liquid SC product lacks pharmacokinetic/pharmacodynamics (PK/PC) assays

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because it is a complex natural product as opposed to a well-defined chemical molecule.

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Thus, controlling product quality faces difficulties [39]. Finally, determining molecular

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structure of these cartilage-derived anti-angiogenic compounds and synthesizing new drugs

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will be beneficial. Of course, bioavailability can be improved by administering drugs in a

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systemic manner.

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reported by Bukowski [36]. Anti-angiogenic effects of SC are illustrated in Figure 1. It

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whether they are valuable in clinical applications or not [40]. Therefore, it is vital to present

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anti-angiogenic drug candidates to standard, clinical, strictly controlled, randomized, and

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double-blinded trials in order to achieve reliable results in the future [4]. On the other hand;

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since angiogenesis can be initiated by numerous angiogenic factors that function via

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distinctive and complex signal pathways, it can be considered as a redundant process. As a

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result, metabolic pathways contributing to angiogenic process can limit the anti-angiogenic

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activity of a single agent; nevertheless, current therapeutic agents fail to change it. In fact,

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better results for cancer treatment can be achieved by combining anti-angiogenic compounds

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with traditional anti-neoplastic drugs in comparison with the use of each product alone [4,

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In addition, effectiveness can be limited by study design and a question might be raised

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different vascularization patterns [4, 42], and some tumors can even get larger without

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angiogenesis [43]. All these factors contribute to the formation of some types of tumors

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resisting against anti-angiogenesis therapy.

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41]. Besides, it has been found that the growth of some types of tumors might follow

3. Immunomodulatory Effects of SCE

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cytokines, and chemokines; contributing significantly to regulation and control of immune

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responses [44]. Evidence has further indicated that producing various cytokines and/or

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chemokines via human peripheral blood leukocytes (HPBL) can be induced by SCEs. Tumor

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necrosis factor-alpha (TNF-α) production is similarly known as the most clearly-described

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cytokine response of HPBL to cartilage stimulation [45]. While TNF-α production is thus

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induced by the extracts of cartilage from calves (i.e. bovine cartilage) and sharks, SC can

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produce higher levels of TNF-α. Moreover; the ability of cartilage extracts from calves (i.e.

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bovine cartilage) and sharks have significantly reduced to induce TNF-α production once

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treated with proteases, trypsin, and chymotrypsin; implying that proteinaceous is an active

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component in cartilage [45]. Besides, SCE stimulates interferon gamma (IFNγ) production at

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levels compared with those obtained in the cultures induced with polyhydroxyalkanoate

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(PHA), as a known mitogen. Therefore; active component behavior in SC is similar to a

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mitogen. It should be noted that leukocytes are in addition stimulated by mitogen to produce

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potent mediators. In addition, it seems that the presence of SC augments PHA activity [45].

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Three potent Th1 type cytokines i.e. TNF-α, IFN-γ, and interleukin 1 beta (IL-1β) are the

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profile of cytokine induction for SC, two of which are particularly contributing to

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Immunomodulators frequently function by inducing particular patterns of growth factors,

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has not been induced by SC [45], so it stimulates a Th1 type response and has an indirect

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inhibitory effect on developing a Th2 response through IFN-γ action, which is the inhibiting

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factor of the expansion of Th2 cell population [46, 47]. Additionally, the pattern of the

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cytokine response expressed in cartilage-stimulated leucocytes has been the final result of the

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responses of successive leukocytes that involve monocytes/macrophages, which first respond

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to SC with the TNF-α release, and are followed by lymphocytes as secondary responders

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producing IFN-γ [14, 15]. However, stimulation of the proliferation of B lymphocyte and

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non-stimulation of T lymphocytes had been observed in a study into the impact of SC

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preparations on purified populations of B cells and T cells [48].

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inflammatory responses and an inflammatory chemokine i.e. IL-8. A significant level of IL-4

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simple SCE with water containing more protein and less proteoglycan (PG) [48]. It is

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noteworthy that nitric oxide, as a free radical with anti-microbial activity, is produced by SC-

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stimulated macrophage. Thus, it can have a potentially important role in increased body

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defense against bacteria.

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Moreover, studies had found that proliferation of mice splenocytes had been induced by

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IL-10, and IL-12. In particular, SC has a strong stimulatory effect on producing IL-6 and IL-

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12. The stimulating capacity to produce certain cytokines by macrophages can also provide

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critical information regarding the main mechanisms contributing to SC immunostimulatory

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In addition, SC can influence macrophage by producing some cytokines such as IL-1, IL-6,

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Th1 and Th2 cell equilibrium, but IL-6 is an inflammatory cytokine [49]. Th1 cells can

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similarly boost cellular immunity as a crucial item for anti-cancer immunity. Therefore,

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immunostimulatory activity is triggered by a complex mixture of protein molecules

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(especially involving collagen) [14, 15].

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The production of RANTES (CCL5) as a chemokine and monocyte chemoattractant protein-1

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(MCP-1/CCL2) by SCE-stimulated peripheral blood mononuclear cell (PBMC) has been

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correspondingly assayed and it has been shown that SC induces multiple-fold higher level of

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MCP-1 in comparison with lipopolysaccharides (LPS). Chondrocytes in cartilage can

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similarly produce MCP-1 in inflamed tissues and induce recruitment of monocytes and

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macrophages [50]. What is more, it is assumed that MCP-1 plays a significant role in RA

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pathology through starting monocyte infiltration.

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Besides, RANTES can have a pro-inflammatory function for T cells and monocytes [51].

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PBMC cultures in both un-stimulated and stimulated environments have additionally

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contained RANTES threshold level, which have not been dissimilar. In spite of this, un-

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activities [48]. It has been revealed that IL-12 is as a natural killer (NK) stimulant that affects

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stimulated cultures might experience a steady decrease of RANTES levels within 96 h, but

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SCE might constantly induce amplified RANTES production. Hence, there is a well-defined

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difference between production of MCP and RANTES in response to SC or LPS leucocyte

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stimulation. This data shows that distinct cell receptors can stimulate these two substances

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and/or it is done by using various activation/signaling pathways [14, 15].

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3.1.

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SC Signaling Pathway

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A significant reduction had been consequently observed in TNF-α production in response to

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LPS when protein kinase C (PKC) signaling pathway had been inhibited, whereas no effect

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Experiments with kinase inhibitors denote distinctions of pathway activation for SC and LPS.

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membrane lipid signaling related to stimulation of LPS, which is lacking when leucocytes are

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stimulated with SC. Thus, SC-stimulated cytokine production does not require PKC. While

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LPS response does not change, phosphoinositide 3-kinase (PI3K) experiences a slightly

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atypical phenomenon, so that inhibition of this pathway boosted cytokine production via SC

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stimulation [14]. With respect to recycling of the receptors to the cell surface, PI3K

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contributes to endosomal trafficking [52, 53]. Perhaps, signaling in response to SC is

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enhanced by blocking PI3K pathway, because the receptor maintains its place on cell surface,

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and its primary activation is augmented. In other words, another regulatory protein may

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had been seen in experiments with SC stimulation [14]. This might be partly caused by cell

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Moreover, mitogen-activated protein kinase (MAPK) signaling facilitates nearly all aspects

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of innate immune responses. Additionally, it is involved in signaling events resulting in

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expressing cytokine gene, particularly for producing inflammatory cytokines and chemokines

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[54]. In order to identify factors engaged in SC stimulation of leukocytes, certain inhibitors

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for different signaling molecules consist of c-Jun N-terminal kinases (JNK), p38 MAPK, and

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other MAPK members (Raf/MEK/ERK). It seems that both JNK and p38 MAPK signaling

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are considered as the essential signaling molecules for TNF-α production in leucocyte

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treatment with SC, while the cytokine production in responding cells is not affected by

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inhibiting Raf/MEK/ERK pathway. In this respect, it has been demonstrated that there are

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numerous possible interrelated signaling pathways activated in response to SC. Furthermore,

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it has been found that almost two of them belong to the family of MAPK [14].

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generally reduce the response of the cells to SC, dominated by PI3K [14].

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3.2.

Immunomodulatory Effects of Dietary SC

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remains the question whether the same immunomodulatory impacts can be induced by dietary

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intake of SC in vivo or not. One relevant answer maybe the degree of cartilage ingestion

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capability to stimulate the intestinal cells, which is dependent on characteristics of cartilage

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fragment consisting of glycosylation pattern, size of fragment after digestion, and level of

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absorption for inducing a systemic impact through circulation [54, 55].

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Since commercial SC retains its cytokine-inducing property in acid extracts, it is concluded

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that the low pH of the stomach will not have an effect on bioactivity. Moreover, SC acid

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extracts can retain 20% of their activity after being treated with digestive enzymes such as

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chymotrypsin and trypsin [45]. There are also various immune cells in the intestine activated

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As demonstrated, SC induces production of cytokine and chemokine in vitro; but there

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exacerbate inflammatory diseases [55, 56]. Furthermore, circulating leucocytes are stimulated

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and they are induced by SC-derived peptides that are adequately small, similar to in vitro

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experiment responses, and can release into circulation through muco-intestinal regions. In

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view of that, immunoregulation can be largely modified if SC fragment enters into blood

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circulation or any region in the body via intestinal absorption. As a result, the health status of

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individuals suffering from inflammatory diseases and more production of Th1 cytokines can

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be affected by cartilage intake and consequently inflammatory cytokine induction may not be

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at a good level. Nevertheless, SC can induce Th1 response and cellular immunity is

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by digested SC protein/peptide, so SC can produce inflammatory cytokine in the intestine and

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whose allergic condition is frequently related to immunoglobulin E (IgE) production because

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Th2 stimulation is absent and IL-4 and IL-10 secretion are limited by SC [14].

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Likewise, in another study, oral intake of keratan sulfate (KS) from SC had established that

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glycosaminoglycan (GAG) could amplify the frequency of Lactobacillus species in the

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intestine, so it could enhance gut microbiota as a new probiotic to treat diseases due to the

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important role of such microbiota in homeostasis and individuals’ health status [57].

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stimulated. On the other hand, SC can be advantageous for individuals with hypersensitivity

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3.3.

Effect of SCE on Endothelial Cells

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Treatment of endothelial cells (ECs) by TNF-α can regenerate the greatest impact of cartilage

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on gene expression profile. ECs treated with SCE (AE-941: a standardized water-soluble

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extract) can experience a higher level of TNF-α, IFN-γ, granulocyte-macrophage colony-

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stimulating factor (GM-CSF), IL-1a, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 [58]. The data

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in the related literature has suggested that changes in gene expression and cytokine

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production have been seen in SC-stimulated ECs inducing reactive oxygen species (ROS)

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enhancer of activated B cell (NF-κB) pathway [58]. It has been also observed that several

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cytokine expressions are induced in SC-stimulated ECs. For this reason, the first proposal is

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that as TNF-α can alone induce an expression profile, almost identical to the one induced by

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SCE, its role may be of importance [58]. Secondly, it is postulated that tumor angiogenesis is

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generally followed by a decreased inflammatory response in vascular endothelium of tumor

240

and these cells may lead to a significant effect [59], so it is assumed that angiogenesis

241

inhibition can restore responses to inflammatory cytokines [60, 61]. However, there are still

242

debates on pro- or anti-angiogenic activity of cytokines.

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It had been similarly demonstrated that NF-κB activation in ECs of bovine aorta could be

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production and hence causing activation of JNK and nuclear factor kappa-light-chain-

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reported that JNK and caspases in bovine aorta ECs could be induced by SCE [62]. Inducing

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a large number of genes by SCE had been also inhibited by pretreatment with N-

247

acetylcysteine (NAC), which could give strength to the viable function of ROS in inducing

248

expression of genes in experiments with SCE stimulation.

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induced by SCE via stimulating phosphorylation of its inhibitory subunit IκB [62]. They had

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and EC detachment via tissue-plasminogen activator (tPA). The increased volume of plasmin

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production induced by SCE can be in this way useful by degrading fibrin matrix, that is

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necessary for developing angiogenesis [63] and other extracellular matrix (ECM) adhesive

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Converting plasminogen (PLG) into plasmin is triggered by unrestrained matrix proteolysis

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vitronectin (VTN) [66]. Final detachment of ECs [67] may significantly improve ROS

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production in cells, which can result in the death of ECs [68].

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Expressing several chemokines such as IL-8 and GM-CSF as well as cell adhesion molecules

257

(CAMs) like selectin E, endothelial adhesion molecule 1 (SELE), and intercellular adhesion

258

molecule 1 (ICAM1) are additionally induced by SCE. In this regard, Dirkx et al. suggested

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that EC anergy could be controlled and interactions of leukocyte endothelium and recruitment

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in tumor sites could be promoted if anti-angiogenesis therapy had been applied [61]. Others

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had also reported that endothelium might experience damage and interference with

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angiogenesis in the case of recruiting leukocyte by chemokines and binding to endothelial

263

molecules [69]. It had been presented in an in vitro study that anti-angiogenic activity of

264

endostatin would require SELE expression [70]. Literature had further reported that

265

endothelial CAMs and leukocyte adhesion could be induced by IFN-γ, IFN-α, TNF-α, and

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IL-1α/β [71]. Additionally, it had been found that guanylate binding protein 1 (GBP-1) had a

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major role in angiostatic function of these mediators. Nonetheless, the immunostimulatory

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glycoproteins such as laminin [64], fibronectin (FN), von Willebrand factor (VWF) [65], and

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function of GBP-1 had been primarily suggested [72]. Recently, it has been established that

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GBP-1 suppresses proliferation of ECs and plays a mediating role in anti-angiogenic activity

270

of cytokine [72, 73, 74].

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4. Low-Molecular-Weight Proteins (LMWPs) of SC

273 274

immunostimulatory properties are mainly associated with its protein content [48]. In

275

consequence, cellular immune response and T-cell infiltration into a tumor may be increased

276

by this fraction [31, 48]. Different studies showed the existence of LMWPs in SC and

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confirmed that most of SC’s biological effects such as anti-angiogenesis activity and

278

increased immune response are related to this component.

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According to the related studies, even though there are many PGs in SCE,

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consisting of 1-10 kDa proteins with the greatest anti-angiogenic function was isolated by

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Oikawa et al. [75]. Moreover, Sheu et al. isolated a potent angiogenesis inhibitor (i.e. U-995)

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with two peptides with 10 and 14 kDa molecular masses [25]. Furthermore, isolation of a

283

fraction composed of 1-500 kDa molecules (i.e. %-941) with anti-tumor, anti-angiogenic, and

284

anti-protease activities was conducted by Dupont et al. In view of that, they reported a

285

relationship between the greatest anti-angiogenic activity and two fractions i.e. a fraction with

286

molecular weight (MW) of ˂100 Da and another one with MW of 8-18 kDa [76]. Chen et al.

287

had demonstrated that growth and attachment of ECs could be inhibited by the extract

288

obtained in their study [77]. Liang et al. had correspondingly reported the maximum

289

inhibitory effect of a fraction with MW of about 10 kDa on the growth of ECs [78].

290

A specific delayed-type hypersensitivity (DTH) skin test had been also utilized to examine

291

certain immune response stimulations by SC. As a result, it had been reported that DTH

292

response had been increased by crude and purified fractions with LMW (14-15 kDa)

293

glycoprotein. Besides, it was easy to generate a DTH response as a cellular immune response

294

marker. Thus, cellular immune response against tumor antigens (Ags) could be boosted by

295

this fraction [13]. Furthermore, the hemagglutination test had been used to determine the

296

impact of crude and purified SC fractions (14-15 kDa protein) on specific humoral immunity

297

and results had revealed that humoral responses had been enhanced by SC, and T cell

298

proliferation could be stimulated by the purified SC (14-15 kDa protein). The animal model

299

had also demonstrated that a growth in CD4+ and CD8+ T lymphocyte infiltration into a

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According to the researchers, tumor angiogenesis is inhibited by SC. For example, a fraction

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that is, SC LMW protein (SCLP) [31].

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As proposed in recent studies, angiogenesis is involved in inflammatory infiltration in

303

malignant diseases [79]. ECs in the site of tumor can apparently have an anergy condition.

304

Besides, there is a poor interaction between these ECs and leukocytes passing through them.

305

Nevertheless, results have indicated that the given anergy may be triggered by angiogenic

306

factors released from tumor cells (that is possibly done by modulating CAMs on ES surface).

307

Therefore, tumor angiogenesis induces EC anergy but angiogenesis inhibition can increase

308

lymphocyte infiltration [80]. In order to achieve an efficient cell-mediated immunity (CMI)

309

response in DTH testing, expression of CAMs facilitating leukocytes arrest is required by

310

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tumor had been induced by intra-peritoneal injection of LMWP of SC (14-15 kDa protein),

ECs in the affected tissues.

311 312

through modulation of CAMs on EC surface [13]. Regardless of the mechanisms of

313

lymphocyte infiltration into tumor tissues, tumor size can be decreased by SCLP. Obviously,

314

this phenomenon is considered as an advantage for tumor therapy. Recent studies have

315

further determined the importance of tumor-infiltrating lymphocyte (TIL) in anti-tumor

316

immune response [13]. T cell infiltration into a tumor is also reduced in tumor progression

317

cases [81, 82]. TIL infiltration (both sub-populations of T-cells: CD4+ and CD8+) is thus

318

greatly encouraged by SCLP fraction. For that reason, SCLP can be used for recruiting T-

319

cells into a tumor [13].

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Cell infiltration and DTH response may be shaped by immunomodulatory fraction of SC

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lymphocytes cells, and inhibiting blood vessels in a tumor can thus predict molecular

322

mechanism of SC in stimulating immune responses and regressing tumor size; otherwise,

323

other unknown mechanisms may be involved. Studies conducted on human breast cancer in

324

stage III patients have consequently denoted that cytokine response in cancer patients has

325

experienced a sharp shift towards Th1 (IFN-γ), while it has been Th2 before treating. Another

326

study on an animal model had shown that homing receptors in tumor lesion after injecting

327

intra-lesion or intra-peritoneal of SCLP had led to increasing ICAM1 on the surface of the

328

subpopulations of CD4 and CD8 T cells [13].

329

Moreover, the greatest immunostimulatory impacts on cytotoxicity of NK cells are induced

330

by SCLP. It has been also suggested that enhancement in cytotoxic performance of NK cells

331

might be seen if there is a direct interaction between SC-derived proteins and NK cell surface

332

receptors [83]; but, another study had demonstrated suppression of NK activity by crude and

333

purified SC [13].

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Shifting cytokine responses, modulating homing receptor of both endothelial and

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cells (DCs) caused by immune strengthening effect of SCLP on tumor treatment and cell-

336

mediated immunity. The impact of SCLP on stimulation and maturation of DCs [12] as the

337

major Ag-presenting cells (APCs) in the immune system was explored in an in vitro study.

338

As confirmed by the higher expressions of MHCII, CD40, and CD86 (i.e. maturation marker

339

of DCs), DC maturation could be induced by SCLP. Up-regulation of co-stimulatory and Ag-

340

presenting molecules similarly denoted DC maturation [12]. It should be noted that an

341

augmented ability of allogeneic T cell stimulation in SC-treated DCs was observed when

342

reviewing the functional properties of DCs. Conversely, this could be totally expected

343

considering the greater expression of co-stimulatory and maturation markers, in particular,

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Accordingly, a correlation may exist between SC and activation of immune cells by dendritic

345

mechanism of DCs activation by SC. SCE aimed to induce cytokines and chemokines in

346

HPBLs were correspondingly investigated by Merly et al. [45], indicating high levels of

347

TNF-α and IFN-γ induced by this extract. These cytokines are popular DC maturation

348

inducers. Therefore, SCLP, which mostly induces DC maturation, can be employed in DC-

349

based immunotherapy of cancer [12].

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MHCII molecules on cartilage-treated cells [12]. Nevertheless, it is necessary to explain the

351

on lymphocytes without the presence of Ag-presenting cells, cartilage protein seems to

352

stimulate and activate through innate immune receptors such as toll-like receptors (TLRs).

353

lP

re

Given that SC has been proved to affect macrophages, monocytes, and DCs but not directly

354

lymphocytes.

355

na

So, SC can have an adjuvant function and induce signals to stimulate and activate T

5. Bioactive Component of SC Proteins

356 357 358

by bone during ossification. It should be noted that ossification does not occur in

359

elasmobranchs and the skeleton is still cartilaginous through adulthood. In principle, cartilage

360

consists of a single type of cell, chondrocyte, and an ECM of mostly water, PGs, and collagen

361

fibers. Besides, PGs are complex macromolecules composed of a core protein, so that GAG

362

side chains are connected to it [84]. Furthermore, there is a relationship between PGs and

363

collagen fibers, which are organized into highly ordered triple helical fibrils and form a

364

network structure of cartilage [85].

365

There are plentiful collagen proteins in cartilage, consisting of three alpha chains. There are

366

correspondingly similar or dissimilar chains in diverse types of collagens which follow a

367

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The skeleton of a vertebrate embryo also contains cartilage, as a connective tissue replaced

11

368

significant types of collagen in extracellular matrix (ECM) of vertebrate cartilage is type II

369

collagen (CII). The glycine repeat characteristic of collagenous areas, observed in many

370

biologically pertinent functional proteins including complement family members and

371

hormones like adiponectin, also exists in CII [87, 88].

372

A study in this domain had correspondingly identified and characterized bioactive

373

components responsible for immunostimulatory activity in commercially prepared SC. It had

374

been revealed that considerable biological activity influencing human leukocyte action

375

related to inflammation had been exhibited by minimum one SC component [89]. In addition,

376

according to the analysis of the protein sequence of derived peptides, the active component

377

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prototypical molecular pattern of Gly-X-Y repeats all over their structure [86]. One of the

378

of collagens are found everywhere in connective tissues of all vertebrates, CII is cartilage-

379

specific, and unlike other collagen types, it apparently represents a major stage in the

380

evolution of cartilaginous tissues since it is limited to vertebrates and does not exist in any

381

invertebrate lineages [87]. However, SC-derived protein seems to depend comparably on its

382

glycosylation pattern as enzymatic deglycosylation leads to complete removal of its cytokine-

383

stimulating activity [89].

384

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-p

had been determined as collagen type II, alpha 1 (CIIa1) protein [89]. Even though majority

385

can function as innate immune ‘‘hazard’’ signals; indicating damage, trauma, and distress of

386

lP

Once released from the ECM following an injury to bones and joints, GAG fragments of PGs

387

Therefore, it is rational to assume that a group of molecules might be represented by

388

cartilage-derived factors actuating Ag-presenting cells, such as tissue macrophages, and

389

resulting in modulating general activity of the cells in such microenvironments that disturb

390

normal physiology at the localized and/or systemic sites [19, 93].

391

Another study had reported that a 37 kDa type-II collagenous peptide (CIIP) extracted from

392

SC could have very strong antioxidant activity and therapeutic applications compared with

393

CII, due to higher content of glycoprotein and hydrophobic amino acids [94].

394

Correspondingly, it has been demonstrated that collagen and type II gelatin derived from SC

395

could have a potent antioxidant effect, assumed to be highly therapeutic [33].

396

Various investigations have reported LMWP in SC ranging from 10 to 37 kDa, which might

397

be due to use of various methods, protein purification materials, or variety of glycosylation

398

and shark species. Overall, as stated in structural and biochemical studies, the nature of these

399

proteins is mostly CII.

400

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tissue and consequently generating inflammatory responses [90, 92] after tissue stress.

401

12

6. Anti-Inflammatory Effects of SC Polysaccharides (SCPs)

402 403

reported in several studies [95]. Proteins, polysaccharides, fat, minerals, calcium, phosphorus,

404

and moisture can be found in SC [96]. SCPs also contain GAGs, such as keratan sulfate (KS),

405

chondroitin sulfate A (CSA), chondroitin sulfate C (CSC), and hyaluronic acid (HA). Several

406

experiments have been conducted on applications of GAGs and chondroitin by millions of

407

patients over the past few years, proving that oral administration of these substances can be

408

useful for patients with joint problems, specifically those with OA. Joint pain can be

409

consequently relieved by polysaccharides. In fact, they can result in rebuilding cartilage and

410

decelerating OA progression [97].

411

ro of

Anti-inflammatory impacts and fibrinolytic functions of GAGs including SCE have been

412

administration of SCPs via suppressing inflammation and joint damage progression,

413

improving radiologic change of bone, accelerating weight loss, and controlling swelling

414

index of the paw. It had been found that the inflammatory process in RA rats without

415

treatment had resulted in significant upsurge in pro-inflammatory cytokines of IL-6 and IL-

416

12. In addition, SCPs had largely inhibited IL-6 and IL-12 over-production [97].

417

As ECM components or soluble molecules in the body, GAGs are linear heterogeneous

418

polysaccharides endowed with an omnipresent expression on cell membrane [98]. One of the

419

main members of the GAG family is chondroitin sulfate (CS), mostly present in cartilage,

420

bone, skin, and blood vessels [98]. It is generally sulfated at chondroitin-4-sulfate (C4S) or

421

chondroitin-6-sulfate (C6S) position and contains repeat disaccharide units of D-glucuronic

422

acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) [98, 99]. One of the sources of CS that

423

seems to be useful in the future is SC [100]. Promising advantages of CS for treating OA

424

have additionally demonstrated its adequate immunomodulatory properties [101]. Producing

425

pro-inflammatory cytokines, ROS, and nitric oxide (NO) is effectively decreased by CS

426

(mainly C4S and C6S) through chondrocytes and synovial cells in vitro [102, 103].

427

Therefore, anti-inflammatory effects of CS will be observed if the expression of p38 MAPK

428

phosphorylation and NF-κB activation in these cells is reduced [104]. On the other hand,

429

studies have shown that bioactivity of some GAGs can be significantly changed through

430

immobilizing. For example; IFN-γ bound to immobilized-GAGs is biologically active,

431

whereas soluble GAGs inhibit its activity [105]. Furthermore, the greatest NO inhibitory

432

activity on macrophage among the GAGs tested has been seen in soluble C6S from SC [106];

433

but, it has been found that NO production by murine macrophage has increased by the CS-

434

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lP

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Research has correspondingly indicated that RA process can be effectively blocked by oral

13

435

macrophage. It has been also found that pro-inflammatory cytokines such as IL-6 and TNF-α

436

production can decrease by C6S in solution through IFN-γ/LPS- or LPS-activated

437

macrophage [106].

438

One of the cytokines needed for inflammation pathogenesis is TNF-α, so that it can induce

439

production of other cytokines such as IL-6 [108, 109]. The antibody blockade of TNF-α has

440

been currently approved by the United States Food and Drug Administration (FDA) due to its

441

capability for treating Crohn’s disease [110] and RA [111]. It has even reached favorable

442

outcomes in diminishing inflammation. In other words, the beneficial biomarker of several

443

inflammation-associated diseases such as psoriasis [112], OA [113], atherosclerosis [114],

444

ro of

immobilized surface [107]. C6S can similarly inhibit NO production by the activated

445

diseases can be expected by the inhibitory effects of C6S on both TNF-α and IL-6.

446

Moreover, the dependence of various impacts of C6S on the production of the pro-

447

inflammatory mediator on macrophage communities had been hypothesized in another study,

448

indicating that the differences among macrophage populations had been found to be

449

important in their cytokine production after stimulating LPS [116]. In particular, studies had

450

demonstrated that C6S-mediated inhibition of macrophage pro-inflammatory phenotype had

451

been also possibly followed by alterations towards anti-inflammatory/wound-healing M2

452

phenotypes, revealed by increased level of IL-10 [106]. The ability to suppress the production

453

lP

re

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and diabetes [115] is IL-6. In a broader context, significant implications for treating these

454

LPS-activated macrophage is thus assumed as one of the common performances of IL-10

455

[117]. Also, the significant role of IL-10 in diminishing uncontrolled inflammatory responses

456

and improving wound-healing has been reported by several animal studies [118].

457

Basically, recent information about mechanisms regulating NO synthesis in macrophage is

458

related to inducible nitric oxide synthase (iNOS) and arginase [119]. In addition, studies have

459

indicated that C6S can somehow change levels of iNOS protein and activity of arginase in

460

LPS-activated and M1 macrophage, respectively, but the amount of changes made has not

461

been adequate to justify anti-inflammatory functions observed in C6S [106]. In contrast,

462

nuclear translocation of NF-κB, as a major transcriptional regulator in inflammation, had

463

been deeply inhibited by C6S during experimental intervals. This showed a possible

464

assumption that NO, IL-6, and TNF-α production had been suppressed by a mechanism

465

called NF-κB pathway. Also, the inhibitory impacts of CS on NF-κB activation had been

466

illustrated by other scholars for chondrocytes [102, 120], synoviocytes [103], and T cells

467

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of NO and pro-inflammatory mediators such as TNF-α, IL-6, and IL-1 by IFN-γ/LPS- or

14

[121] to support this observation [106]. It should be noted that there are at least two ways, in

468

which NF-κB nuclear translocation can be affected by C6S:

469

(i) After establishing an interaction between C6S and their specific receptors on the

470 471

macrophage, a negative signal may be delivered, (ii) C6S may act as an antagonist, interfering with LPS binding to membrane receptors

472 473

(e.g. TLR4) on macrophage [106].

474

stimulated chondrocytes [120]. These findings were consistent with the latter hypothesis.

475

However, experiments are being conducted to further explore and test this study hypothesis.

476

In one study, different LMW chondroitin sulfates (LMWCSs) purified from three sources of

477

ro of

It has been confirmed that C6S can be interrelated with TLR4 receptor complex in LPS-

bovine, porcine, and shark along with their effects had been investigated in a mouse model of

478

OA. So, LMWCS prepared from SC via oxidative degradation method had inhibited and

479

regulated alternative complement pathway and had prevented chondrocyte cell death leading

480

to a reduction in OA symptoms [122].

481 482

decreased expression of inflammatory mediators including TNF-α, IL-1β, matrix

483

metallopeptidase 13 (MMP13), nitric oxide synthase 2 (NOS2), IL-10, and cyclooxygenase-2

484

(COX-2). It had also preserved the activity of antioxidant enzymes including superoxide

485

dismutase (SOD) and catalase (CAT) in the joints. Mass spectroscopy analysis had further

486

lP

re

-p

Likewise, oral administration of PG in OA mouse model had improved symptoms and had

487

whose sequence resembled the core protein of aggrecan and epiphycan [123].

488

7. Conclusion

na

revealed two unique peptides i.e. AGWLSDGSVR and LDGNPINLSK in the purified PG,

489 490 491

example, its anti-cancer effects are regarded as the most important reason for its marketing

492

[1, 5]. Anti-angiogenic and anti-tumor activities of SC have been demonstrated in animals

493

and humans [6, 8]. Scientific evidence has correspondingly suggested that SC induces

494

stimulation of the immune system and has anti-angiogenic impacts, leading to its

495

effectiveness against tumor progression, as well as bacterial, viral, parasitic, and fungal

496

infections [45, 124]. Besides, it has been found that SC LMWP is the most potent fraction

497

enhancing immune response [13]. In vitro studies have further indicated that SC is composed

498

of LMWP specially ColIIa1 protein and this fraction of SC can stimulate the immune system

499

via induction of Th1-type inflammatory cytokine [89]. Moreover, cytokines and ROS might

500

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ur

Special characteristics of SC have conventionally brought about medical advantages. For

15

501

CAMs can consequently induce the recruitment of immune cells to the developing

502

endothelium [58] and angiogenesis can be inhibited by SCE. Therefore, bioactive

503

components exist in dietary SCE. Immunoreactive cells are correspondingly stimulated by

504

these bioactive components for producing inflammatory cytokines and chemokines.

505

Likewise, the components can trigger specific paths of signaling for immunostimulation.

506

On the other hand, anti-inflammatory agents of SC are CS in vitro and in vivo [125].

507

Decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokine IL-10 thus

508

represent CS-C modulatory activities from SC [106]. Furthermore, evidence has supported

509

that immunomodulatory activities have been changed by immobilizing specific GAGs (e.g.

510

ro of

play a role in activating the early ECs by SCE. Secretion of chemokines and expression of

511

murine macrophages in several ways from soluble GAGs. In addition, there is additional

512

evidence confirming anti-inflammatory and protective roles played by soluble C6S [106].

513

Therefore, it is concluded that immunostimulation and angiogenesis inhibition for cancer

514

therapy are induced by the protein fraction of SC, especially LMWP. Yet, the anti-

515

inflammatory response is induced by PG and CS which are useful for inflammatory diseases

516

including RA and psoriasis. Immunomodulatory effects of different fractions of SC

517

preparations are summarized in Table 1.

518

re

-p

C6S, HA, and HS), so that the production of IFN-γ/LPS-induced NO have been influenced by

lP

On the other hand, studies have shown that both SC protein and polysaccharides have strong

519 520

diseases. Besides, inflammation in cancer microenvironments can have side effects and

521

induce tumor progression; therefore, scientists make attempts to decrease inflammation in the

522

site of tumor. It can be stated that the effects of whole SCE, containing both protein and

523

polysaccharide fractions, can be applied to a wide range of diseases.

524

However, at present, SC is being used in the form of a supplement with multiple side effects

525

such as nausea, vomiting, abdominal cramps and/or bloating, constipation, hypertension,

526

general malaise and hypercalcemia and it has not even received the FDA approval. With

527

respect to implications for consumers and the environment, identifying the active components

528

of this natural substance and synthesizing it or using other alternative sources can reduce the

529

consequences and even augment the efficiency of this supplement to obtain FDA approval for

530

certain diseases such as cancer and inflammatory diseases. With advances in science and

531

given the linkages between biochemical and cellular processes in the human body, such as

532

the connection between angiogenic pathways and the immune system, various effects of

533

substances can be better analyzed and then used for diseases. Considering the function of

534

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antioxidant effects that are very useful and applicable in both cancer and inflammatory

16

effective SC molecules, it is suggested to reflect on functions, mechanisms of action, and

535

synthetic production of these materials.

536 537

Declarations of interest: none

538 539 540

Conflict of Interest

541

Not declared.

542 543

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Figure1: Mechanisms of SC anti-angiogenic effects. SC induces inflammatory cytokine production and these mediators cause endothelial CAM expression and thus immune cell infiltration to tumor site and also endothelial cell apoptosis. However, GBP-1 can be induced by inflammatory cytokine and inhibit EC proliferation. Other mechanisms of anti-angiogenic effect of SC are MMP inhibition, VEGF-VEGFR binding inhibition, βFGF inhibition and tPA induction, angiostatin and collagenolysis by collagenase and degradation of fibrin matrix. VEGF: vascular endothelial growth factor; βFGF: basic fibroblast growth factor; MMP: matrix metalloproteinase; tPA: tissue-plasminogen activator; CAM: cell adhesion molecule; GBP-1: guanylate binding protein 1; EC: endothelial cell

827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862

863 864

23

Table 2: Immunomodulatory effect of different component of shark cartilage Preparati on

Cell

SCE

HPBL

Effect

Method/

Result

865

Refrences

Mechanism TNF-α, IL-1β

14, 44, 49

JNK and p38 MAPK signaling

IFN-γ, IL-8 MCP, RANTEX IL-1β,IL-6,

Macrophage

47

TNF-α, IFN-γ,

ROS and caspase production,

GM-CSF, IL-1a,

IL-8, IL-, IL-12

DTH test

Cellular immune response

lP

(CIIa1)

re

(SELE, ICAM1), tPA T cell

57, 61

-p

Leukocyte recruitment

Adhesion molecules

SCLP

Endothelial cell anergy and apoptosis

NF-κB, JNK signaling,

IL-2, IL-4, IL-6, EC

ro of

IL-10, IL-12

TIL infiltration

13, 30

in to tumor site Tumor size decrease

ICAM 1

Jo

ur

NK cells

Humoral immune Hemagglutination responses test

na

B cell

DC

Cytotoxicity

13

82

MHCII,

Maturation

CD40,

marker

CD86

24

12

PG

Macrophage

TNF-α, IL-6, IL-1

M2 induction

NF-κB

(C6S)

Joint pain relieved,

105

Rebuilding cartilage, decelerating osteoarthritis and progression blocked

iNOS, arginase IL-10

SCE: Shark Cartilage Extract, HPBL: human peripheral blood leukocytes, EC: endothelial cells; tPA: tissue-plasminogen activator; SCLP: Shark Cartilage Low molecular weight Protein, DTH: delayed-type hypersensitivity; DC: Dendritic cell, CIIa: collagen type II, alpha 1; PG:

Proteoglycan; C6S: chondroitin-6-sulfate.

866 867 868 869

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870

25

871 872 873 874