Salmon Nasal Cartilage Proteoglycan: Exogenous Functionality Compared to Other Chondroitin Sulfates

Abstract

Chondroitin sulfate (CS), a glycosaminoglycan (GAG) member, is a partially sulfated linear polysaccharide. CS covalently binds to core proteins to form chondroitin sulfate proteoglycans (CSPGs), which are ubiquitously distributed on the cell surface and within the extracellular matrix. Over the past two decades, CS/CSPG products derived from natural sources, such as bovine, porcine, and salmon, have garnered increasing attention worldwide as promising agents for cosmetics, pharmaceuticals, and nutraceuticals. This review explores the characteristics of salmon cartilage proteoglycan (sPG) in pharmaceutical and nutraceutical oral administration compared with other CS/CSPGs. sPG can be easily extracted as a safe, functional anti-inflammatory and analgesic agent. sPG and other CS/CSPGs are translocated outside the gastrointestinal tract after oral intake. However, the translocated amount is minimal, and the underlying mechanism remains unclear. In addition to the conventional narrative that CSPGs exert their effects after absorption or translocation, this review discusses the potential behavior of CS/CSPGs that are not absorbed and remain in the gastrointestinal tract. The interaction activity of the CS chain and regulatory effects of sPG on intestinal bacterial flora suggest the potential luminacoid nature of CS/CSPGs. These insights improve our understanding of the functionality of exogenous CSPGs and expand their potential therapeutic applications.

1. Introduction

1.1 Structure and function of chondroitin sulfate (CS)/chondroitin sulfate proteoglycans (CSPG)

Glycosaminoglycans (GAGs) are linear polysaccharides comprising repeating disaccharide units. Each disaccharide unit consists of an amino sugar moiety (hexosamines, such as D-glucosamine and D-galactosamine) alternately paired with a uronic acid moiety (D-glucuronic or L-iduronic acid). GAGs are classified into four major groups based on their disaccharide components: hyaluronic acid (HA), CS, heparan sulfate (HS), and keratan sulfate (KS). Except for HA, all GAGs covalently bind to core proteins to form diverse proteoglycans (PGs). Endogenous PGs are widely distributed on the cell surface and within the extracellular matrix (ECM) of all cell types, where they cooperate with other glycoconjugates to form the glycocalyx. Many PGs play critical roles in various biological and pathophysiological processes.

CS is the most abundant GAG found in the human body. The disaccharide unit of CS consists of N-acetyl-D-galactosamine (GalNAc) as hexosamine and D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) as uronic acid[1, 2]. IdoA is synthesized through the epimerization of GlcA, a reaction catalyzed by the dermatan sulfate epimerase enzyme family. When IdoA is present at a proportion substantially greater than that of GlcA, the compound is referred to as dermatan sulfate (DS). However, CS and DS regions often coexist within a single chain, referred to as CS/DS. Mizumoto and Yamada provided a detailed discussion on the relationship between CS and DS, and the biological significance of DS [3]. The sulfate groups are randomly esterified on the hydroxyl groups of the CS/DS chains, resulting in various classes, such as GlcA-GalNAc4S (CS-A), GlcA-GalNAc6S (CS-C), GlcA2S-GalNAc6S (CS-D), GlcA-GalNAc4/6S (CS-E), and non-sulfated GlcA-GalNAc (CS-O). The CS structure exhibits considerable heterogeneity owing to the diverse arrangements of these disaccharide units.

The biological functions of CS are strongly associated with its structure, particularly the sulfation pattern. In neurophysiology, a substantial amount of CS exists in the central nervous system as a covalently bound proteoglycan (CSPG) moiety. Although disulfated disaccharides, such as CS-D and CS-E, constitute only a minor proportion, they have notable functional significance [4, 5]. Sakamoto et al. [6] demonstrated that only CS-E residue-rich short stretches, rather than other CS domains, bind to receptor-type tyrosine phosphatase S (PTPRσ) in competition with HS. This binding disrupts autophagic flux, leading to the formation of dystrophic endoballs and the inhibition of axon regeneration. In contrast, Shida et al. [7] reported that CS-D residues facilitated neurite extension via integrin αVβ3-mediated signal transduction, whereas CS-C residues did not exhibit this effect. These findings indicate that the biological activity of CS is highly dependent on its sulfation pattern.

1.2 Exogenous CS/CSPG usage (cosmetic and oral agents)

In addition to the accumulating studies on endogenous CS and CSPG (CS/CSPG), the exogenous use of CS/CSPG has attracted increasing attention. Exogenous CS/CSPG use ranges from cosmetics to pharmaceuticals and supplements. As a cosmetic agent, CS/CSPG is believed to have moisturizing effects and control skin conditions. The precise biological mechanism underlying these effects remains unclear; however, the water-retention properties of CS may have moisturizing effects. The epidermal growth factor (EGF)-like region in the C-terminal G3 domain of aggrecan and versican has been suggested to act as an EGF ligand [8, 9], and skin maintenance effects, including wound healing, may be due to this activity. In addition, CS/CSPG may have an anti-ultraviolet (UV) effect [10, 11], which may be due to its antioxidant properties. As an orally administered agent, including pharmaceutical products and nutraceutical supplements, CS/CSPG is expected to have analgesic and anti-inflammatory effects against joint pain caused by conditions, such as osteoarthritis (OA). In Europe and the USA, CS is authorized as a pharmaceutical product and a symptomatic slow-acting drug for OA (SYSADOA) [12, 13, 14, 15, 16, 17, 18]. CS has been marketed as a nutraceutical supplement in other countries, particularly Japan, claiming similar effects.

However, the precise mechanisms and behaviors of CS/CSPG after oral administration remain unclear. At least some absorption or translocation of CS is expected to occur in the proximal small intestine [19]. CS chains generally have a high molecular weight, therefore, it is hypothesized that the paracellular, rather than the transcellular pathway, is dominant in their transport by epithelial cells [19, 20]. However, the extent of the translocation is limited. In addition, the contribution of intestinal bacterial flora is related to CS behavior.

1.3 Brief characteristics of salmon nasal cartilage proteoglycan (sPG) and overview of this review

sPG is the major CSPG product marketed in Japan. The CS used in laboratory studies is mainly derived from mammals, such as bovine, porcine, and whale. These materials have problems with religious restrictions and serious infectious diseases (e.g., bovine spongiform encephalopathy); however, using salmon can avoid these problems. Salmon has a long history of consumption worldwide, which ensures its safety for oral consumption. Salmon nasal cartilage is generally discarded. However, in Japan, it is referred to as hizu, a traditional food. Majima et al. developed a method for extracting proteoglycans from salmon (Oncorhyncus keta) nasal cartilage using acetic acid at a concentration similar to that of vinegar [21]. This method is extremely simple compared with previous proteoglycan extraction methods, which are complex and involve multiple steps. It was the first in the world to achieve the mass production of proteoglycans. Acetic acid extracts from salmon nasal cartilage are composed mainly of aggrecan, a representative CSPG [22]. Kakizaki et al. [23] obtained microscopic images of the molecules using atomic force microscopy (AFM), clearly showing the test-tube brush-like structural characteristics of aggrecan.

Notably, sPG is the only proteoglycan administered to animals and cells as a CSPG, rather than as a CS single chain [24]. As mentioned above, sPG has a typical proteoglycan-like conformation. This conformation and densely packed nature of CS may produce results different from those obtained when CS is administered alone. Anti-inflammatory, cell proliferation, anti-angiogenesis, anti-arthritic, anti-diabetic, and wound-healing effects have been reported [10, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35]. However, detailed molecular mechanisms underlying these effects remain unclear. When administered orally, the same absorption or translocation mechanisms as those of the other CS/CSPG products mentioned above remain unresolved.

In this review, the effects and mechanisms of sPG administration reported to date are summarized and compared with those of CS/CSPG obtained from non-salmon sources, and the mechanisms underlying these effects are discussed. In addition, the possible but uninvestigated effects of orally administered sPG are discussed based on information on the structure of sPG and the physiological activity of other CS/CSPG studies. In particular, based on the fact that CS/CSPG is a non-digestible component that reaches the distal section of the intestine without the influence of digestive enzymes, and interaction of CS/CSPG with membrane and ECM proteins known to exist in the intestinal lumen, the possibility that CS/CSPG exhibits luminacoid behavior.

2. Structural characteristics of salmon nasal cartilage proteoglycan

Salmon nasal cartilage proteoglycans were derived by an extraction method using a low concentration of acetic acid [21]. The fractions extracted by this method contained aggrecan and epiphycan [23, 36]. Notably, sPG are often a mixture of proteoglycan species. Aggrecan is a member of the hyalectan family, and the N-terminal G1 domain of these proteoglycans has a common non-covalent hyaluronic acid-binding nature [37, 38, 39]. The binding of the HA and hyalectans is stabilized by link proteins, and lower pH conditions disrupt this binding because the electric dissociation of the carboxyl groups on HA is a cue for binding. Therefore, the acetic acid extraction method is considered to be a method to release proteoglycans into solution by inhibiting the association of HA, hyalectan, and a link protein.

The molecular weight of sPG was determined to be 344,000 using SDS-PAGE [22]. This value is small for cartilage proteoglycans. AFM images obtained by Kakizaki et al. [23] show a small test-tube-brush-like molecule. The disaccharide composition of the CS chain on sPG was CSA/C/D/O=28/58/7/8.6 [21, 22, 23]. This CS-C-rich and CS-D-containing composition is similar to that of shark-fin cartilage CS. In contrast to monosulfated disaccharide units, which are often having no notable insignificant, disulfated disaccharide units regulate the activity of the CS chain [7, 40]. The CS-D units in the CS chain of sPG are also crucial for proteoglycan function. In addition, aggrecan in sPG is free of KS [22]. KS contamination is a frequent quality control issue in CS products worldwide and [12, 18, 41 42, 43, 44, 45, 46, 47] may be avoided in sPG products. sPG have been studied in many fields. See Table 1 for a summary of previous sPG studies.

Table 1: Research on the effects of salmon nasal cartilage proteoglycan

Species	Dosage	Oral Admin-istration	Results	Ref.
Anti-inflammatory effects
Mice (RAW264.7 cell)	50–500 µg/well	-	Heat-killed E. coli cells-induced TNF-α production was suppressed. STAT3 phosphorylation and expression were enhanced.	[27]
Rats (Wistar rats)	1% PG, 1.5 mL	Yes	Clinical symptoms of experimental colitis and leukocyte number were improved. Total SCFA and n-butyrate in feces were enhanced.	[48]
Mice (C57BL/6)	0.08–2 mg/mL 100 µL/day, 30 days	Yes	Clinical/histological severity of experimental autoimmune encephalomyelitis was improved. Foxp3 expression in CD4+CD25+ cells was enhanced.	[30]
Mice (C57BL/6)	1 mg/day	Yes	Papain-induced allergic response was suppressed.	[31]
Anti-arthritis effect
Human	10 mg, 16 weeks	Yes	Clinical symptoms of knee joint discomfort in subjects were improved.	[35]
Human	10 mg, 12 weeks	Yes	Mild knee pain in subjects was improved.	[49]
Wound healing/Cell proliferation promoting effect
Mice (NIH3T3, TIG-118 and TIG-121 cells)	0.1–1000 µg/mL	-	CD44-dependent wound healing was enhanced.	[33]
Mice (BALB/c)	0.4–10 mg/mL 10 µL/day, 14 days	No	The early stage of wound healing was enhanced.	[28]
Human (NHDF cells)	100–1600 µg/mL	-	ERK-driven Cell proliferation was enhanced.	[29]
Mice (C3H/HeN)	Approx. 1mg/day	Yes	Hair growth after depilation was enhanced.	[50]
Intestinal bacterial flora regulatory effect
Mice (C57BL/6)	10 mg/mL 100 µL/day, 30 days	Yes	The numbers of probiotic and immunoregulation-related bacteria were enhanced.	[51]
Metabolism regulatory effects
Rat (jejunum sacs)	1.5–10 mg	-	SGLT1-dependent glucose transport was suppressed.	[34]
Mice (ICR)	1 mg/day	Yes	High-fat diet-induced increased body weight, serum lipids, and SREBP-1 mRNA levels were suppressed. Leptin level in blood was regulated.	[32]
Anti-angiogenesis effect
Human (EA.hy926 cells)	0.02–1.0 mg/mL	-	The tube formation was suppressed.	[25]
Chick (fertilized eggs)	10–80 µg	-	The formation of blood vessels in the chorioallantoic membrane was suppressed.
Skin condition maintenance effect
Human (HaCaT, NHDF cells)	5–25 µg/mL	-	The skin barrier function was maintained even in UVB-induced damage conditions.	[10]
Mice (hairless SKH-1)	1 or 5 mg/kg/day	Yes

3. The immunoregulatory activities of sPG

3.1 Direct effects of sPG

Immunoregulatory activity of sPG was previously demonstrated. In vitro, the administration of sPG decreased the stimulation of RAW 264.7 cells by heat-killed Escherichia coli cells [27]. TLR4 production was also reduced, suggesting the suppression of signal transduction by the lipopolysaccharide receptor. Pro-inflammatory factors, such as tumor necrosis factor (TNF-α) and inducible NO synthase (iNOS), were simultaneously suppressed. Another study with differentiated Caco-2 cells [52] also indicated the suppression of TNF-α induced interleukin-6 (IL-6) production by sPG, suggesting the inhibition of TNF-α driven inflammation. In vivo, sPG reduced the bacterial number and elevated anti-inflammatory cytokine, (TGF-β and IL-6) production in a Staphylococcus aureus-infected wound [28]. Oral administration of sPG reduces cytokine expression in lamina propria monocytes in an experimental mouse model of colitis [30]. Therefore, sPG exerts an immunoregulatory effect by modulating the expression and activity of cytokines. However, the detailed pathways involved have not yet been identified. Upregulation of Foxp3+ T cells and induction of the expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) [30] were shown by sPG administration, suggesting that sPG possibly has an anti-inflammatory effect by inducing the differentiation of regulatory T cells (T_reg). Foxp3, forkhead box protein p3, is a differentiation marker of T_reg. The mechanisms underlying T_reg differentiation and CS administration are unclear. Only a few studies have shown that sea cucumber-derived CS (fucosylated CS) affects T_reg differentiation [53]. The contribution of the CS chain structure is unclear; however, the effect of sPG on T_reg cells may be due to the CS chain.

Regarding the immunomodulatory effects of CS, several in vitro studies have shown that an anti-inflammatory effect mediated by the promotion of nuclear translocation of NF-κB is becoming a common explanation [14, 16, 54, 55, 56, 57, 58]. Hsu et al. [58] reported the effects of CS on the proliferation and migration of chondrocytes (chon-001). CS administration decreases the phosphorylation of Akt, IκB kinase, and IκB. This leads to the inactivation of NF-κB, a downstream factor, and consequently, to the suppression of matrix metalloproteinase (MMP) family expression. MMP family proteins are IL-1β-induced collagen-degrading enzymes and are significant causes of the disruption of articular cartilage structures in OA lesions [59, 60, 61]. Increased expression of metalloproteinase inhibitor-1 and -2 (TIMP-1 and -2) also suggests the functional inhibition of MMP family proteins. Modulation of the NF-κB axis and expression of TIMPs alters the functional profile of the MMP family and interferes with the destruction of the chondrocyte ECM [58, 59, 60]. In addition, the expression of type II collagen increases, thereby improving the state of the ECM. IL-1β stimulation was downregulated in the presence of CS. The inhibition of IL-1β induced NLRP3 inflammasome activation has also been observed [54]. It is unclear whether this downregulation is mediated by blocking IL-1β receptors or the sequestration of cytokines through the interaction of CS and IL-1β. Potential interactions between sPG and hematopoietic cytokines have also been observed. HS, a member of the GAG family that shares a common structure with CS, is well-known for its ability to bind cytokines and morphogens [65, 66, 67, 68]. The CS chain, especially the CS-E units-abundant chain, exhibits activities similar to those of HS [69, 70, 71, 72, 73]. The structure-dependent binding of the CS chain to immunomodulatory factors, including interleukin, needs to be studied. Moreover, this explanation using NF-κB-related anti-inflammatory signaling is limited to systems where sPG can directly reach affected areas and cells. The systemic effects of orally administered CS/CSPG are discussed in the next section.

3.2 Systemic effects after oral sPG administration

Oral administration of sPG to mice with papain-induced experimental respiratory disease decreased the titer of serum IgE, infiltration of eosinophils, and epithelial or T helper type 2 cells-related cytokines [31]. In a collagen-induced allergic model of rheumatoid arthritis, the clinical and histological severity of the disease and pro-inflammatory cytokine production in the joints were reduced [26]. These experiments demonstrated the cytokine modulatory effects of orally administered sPG in affected areas, far from the gastrointestinal tract. In addition, clinical studies have revealed sustained improvement in diagnostic scores with sPG administration, especially in patients in poor conditions [35, 49, 74]. sPG has also improved collagen degradation and cartilage metabolism markers in the serum and urine, suggesting inhibition of cartilage degradation or promotion of synthesis. These results indicated that oral administration of sPG may exert anti-inflammatory and analgesic effects that are not solely dependent on its cytokine-modulatory effects. However, the amount of CS absorbed by oral administration is limited in many cases. Therefore, whether sufficient amounts of sPG reach the affected areas in these experiments, such as the respiratory organs and joints, is uncertain.

In addition to salmon, proteoglycans from sharks have also been investigated for their oral effects. A fraction of cartilage from the bramble shark (Echinorhinus brucus) was extracted by Ajeeshkumar et al. [75], which appeared to contain aggrecan and epiphycan. The bramble shark PG oral administration at doses of 50 mg/kg or higher improved the histopathological characteristics of the monosodium iodoacetate-induced OA rat model. The mRNA levels of pro-inflammatory cytokines TNF-α, IL-1β, MMP-13, NOS2, and COX-2 were reduced in the joints, while the anti-inflammatory cytokine, IL-10, was enhanced. Kitahashi et al. [76] prepared a CS/protein mixture from blue shark (Prionace glauca) and orally administered it to experimental pancreatic cancer mouse models. Although the mechanism is unknown, serum MMP-9 inhibitory activity was significantly enhanced after oral administration. The authors hypothesized the importance of the protein moiety, based on the lack of MMP-9 inhibitory activity of purified shark CS. However, according to the literature described above, this can be considered as an effect of the MMP inhibitor induced by the CS moiety. These results suggested that sPG and other CS/CSPG products exhibit systemic effects after oral administration. In Figure 1, the potential activities of CSPGs in the level of intracellular signaling that we have mentioned above are briefly summarized.

Figure 1: Potential effects of CSPG on cellular signal transductions. CSPG suppresses NF-κB axis-induced inflammation and ECM degradation. Conversely, CSPG enhances cell proliferation via ERK1/2 activation; however, the upper-stream receptors are not being identified. Solid and dotted arrows indicate activation and nuclear translocation, respectively. The numbers in the CS structure shown in green indicate the position of the sulfate group.

As mentioned above, many in vitro studies have shown that the direct exogenous administration of CS to chondrocytes can contribute to homeostasis by suppressing the inflammatory state of chondrocytes and inhibiting the destruction of aggrecan and collagen. These in vitro studies have been used as an explanation for the systemic clinical effects of oral administration. However, in interpreting the results of these studies, it must be noted that CS is an originally endogenous factor in the ECM of most somatic cells, including chondrocytes [2, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87]. CS synthesis and homeostatic alterations affect phenotype, proliferation, and migration of various cell species. Exogenous CS can substitute for some of the functions of endogenous CS. Therefore, experiments in which CS is administered directly to cells merely observe the results of altering the ECM of cultured cells, rather than reproducing the results of CS reaching the affected area after oral intake. Notably, many of them in vitro studies administered fairly large doses of CS, as described by Henrotin et al. [13]

CS is recognized as a slow-acting drug and some studies suggest that it may take up to two years of dosing to achieve benefits [19, 88]. Continuous use may lead to accumulation. However, accumulation has not yet been traced clinically or experimentally. Therefore, it is crucial to quantitatively study the distribution of CS in the body after oral administration.

4. Behavior of CS/CSPG after oral administration

Only Kudo et al. [52] reported experiments to trace the degradation of orally administered sPG in mice, showing that while the core protein underwent digestion, the CS moiety was not degraded in the digestive tract, at least up to the small intestine. Stellavato et al. [18] reported that CS might reach the intestine with little loss in digestion studies mimicking the stomach and intestine. To date, the only human extracellular enzyme capable of degrading CS chains is the hyaluronidase family, particularly hyaluronidase-4 (HYAL4) [89, 90, 91, 92]. Since HYAL4 expression is not ubiquitous and is spatiotemporally localized, it does not contribute to CS degradation in the digestive tract.

Barthe et al. [93] performed in vitro transit and digestion studies on 14C-labeled CS, and found that degradation occurred only when it was mixed with the contents of the colon and cecum. Absorption occurred in small amounts in the small intestine and, to a greater extent, in the colon and cecum. However, the only component absorbed was the disaccharide. Another study reported that CS did not undergo any degradation in the gastrointestinal tract other than by intestinal bacteria and absorption of CS is thought to occur in the proximal portion of the intestine [18].

Several radioisotope studies [94, 95, 96] in healthy adults have shown that orally administered CS is distributed in the liver, kidneys, and urine. Volpi [97, 98] revealed that the concentration of high-molecular-weight CS (leaving at least 10 residues) in the blood of healthy adults increases after its oral administration. These results suggest that orally administered CS can translocate from the gastrointestinal tract into blood vessels. However, the amount of CS absorbed or translocated is extremely limited. In Volpi’s experiment [97], in which 4 g of Chondrosulf^®, derived from bovine, was administered to healthy volunteers, the amount of CS in the blood was 263% of baseline with endogenous CS. In another study by Volpi [98], in which 4 g of fish-derived CS was administered, blood CS levels increased by 126%. In contrast, Jackson et al. [99] could not detect CS transfer into the blood. This is because the amount of endogenous CS in the blood is very low (around 4 µg/mL), making detection and comparison difficult. Therefore, the amount absorbed by Volpi in these experiments was also insignificant.

Adebowale et al. [46] used monolayer-cultured differentiated Caco-2 cells as a model of the human intestinal epithelium and investigated the export of CS from the intestinal lumen via the paracellular pathway of the intestinal tract. Explanations based on the paracellular pathway have garnered attention. However, the size of the CS that can be transported via the paracellular pathway is extremely limited, even in vitro [20]. CSPG is much larger than a single CS chain, therefore, translocation through the gastrointestinal epithelium is more difficult for CSPG than for CS.

Preliminary ex vivo experiments using everted sacs have also been reported [100], in which dose-dependent sPG absorption was detected in the jejunum, ileum, and colon of the rat intestine. Addition of an inhibitor of clathrin-mediated endocytosis inhibited sPG absorption. In this preliminary study, although the setting of the control group in the inhibitor experiment was inappropriate, the decrease in detection from the proximal to distal supported CS/CSPG absorption in the proximal small intestine.

Therefore, the amount and molecular weight of CS that can be absorbed after oral administration are limited. CSPG, such as sPG, are even more limited because they are larger than a single CS chain. In addition, because the CS chains are rarely digested in the proximal small intestine, which is a possible site of CS absorption, the low-molecular-weight fraction originally present in the product would be absorbed rather than the depolymerized CS derived from digestion. Therefore, most CS/CSPGs would appear to pass through the gastrointestinal tract as passengers.

5. What can CS/CSPG do without being absorbed

The behavior of orally administered CS is not straightforward [15, 99]. As mentioned previously, CS intrinsically contributes to several physiological processes in various cell types. This suggests that CS interacts with signal transduction-associated proteins. Integrins, receptor type kinases, and N-cadherins are candidates [7, 29, 40, 101, 102]. When sPG was administered to normal human dermal fibroblast (NHDF) cells, a cell proliferation-promoting effect with increased ERK1/2 phosphorylation was observed, suggesting the contribution of integrin signaling [29]. These interactions indicate that part of CS/CSPG, which is not absorbed after oral administration and is considered a passenger, can still act as a messenger, potentially interacting with cells that form the lumen of the gastrointestinal tract.

Galectin-3 (Gal-3), a multifunctional lectin, is present in healthy intestinal epithelium [103]. An interaction between the CS chain and Gal-3 has been previously reported despite its physiological significance remains unclear [104]. The importance of Gal-3 and CS, particularly in the cancer microenvironment, has been independently reported [105, 106, 107, 108]. However, the relationship between Gal-3 and CS in healthy tissue requires further investigation. Furthermore, integrin αVβ3 is a candidate receptor protein of the CS-D units-rich CS chain in the central nervous system. Integrin αV is expressed in macrophages [109], suggesting that macrophages in intestinal lymph nodes may potentially receive CS chains. sPG contains approximately 7% CS-D, therefore, it is expected to be trapped by integrin αV.

Pectin is an example of a polysaccharide that interacts directly with proteins in the intestinal tract. Pectin is a polysaccharide obtained from land plants and a typical dietary fiber [110, 111]. Pectin influences the behavior of animal cells, including proliferation and differentiation [112, 113, 114, 115, 116, 117]. Pectin has direct nonprebiotic and prebiotic effects on the intestinal tract [112, 118, 119]. Pectin is associated with fibronectin, and the association of fibronectin with integrin β1 is also disrupted by the presence of pectin [120]. These properties may explain the physiological effects of pectin. Notably, the interaction between CS and fibronectin is classically studied well [39, 121, 122, 123]. CSPG binds to fibronectin and collagen in a CS chain-dependent manner and HA in a core protein-dependent in joints, forming a network structure that makes joints more resistant to mechanical impact. This similarity between the two carboxy group-rich acidic polysaccharides implies the potential interaction of CS in the gastrointestinal tract.

Finally, CS is utilized by the intestinal bacteria. CS is degraded by intestinal bacteria to produce hydrogen sulfide (H₂S). Oral administration of CS increases levels of H₂S and its producing bacteria; glucagon-like peptide 1 (GLP-1) and insulin secretion; and glucose tolerance [124, 125, 126]. These alterations may have been caused by H₂S. Presumably, the sulfate groups of CS are a good source of sulfur for intestinal bacteria. Oral administration of sPG decreases mRNA levels of SREBP-1 in the liver [32]. H₂S regulates the expression of SREBP-1, therefore, oral sPG administration is considered to influence SREBP-1 via H₂S production. Oral administration of sPG has also been reported to modulate the intestinal microbiota and increase the amount of short-chain fatty acids [48, 51]. These results indicate that CS is a prebiotic agent.

Thus, the amount of CS/CSPG absorbed and distributed throughout the body is insignificant. CS/CSPG distributed in the body may not only act directly on the affected area but also the whole body. It is possible that unabsorbed CS/CSPG interacts with proteins in the gastrointestinal tract and acts prebiotically. The function of CS/CSPG may be exerted not only by absorption, but also by other pathways in the gastrointestinal tract. The state of CS/CSPG in the gastrointestinal tract remains unexplored, but it potentially exhibits dietary fiber or luminacoid behavior, similar to pectin. sPG is larger than CS alone, and is expected to have poorer absorption. Studies focusing on luminacoid-like behavioral properties and absorption are needed to elucidate the effects of oral administration of sPG.

6. Conclusions

sPG is a safe proteoglycan with no concerns regarding religious restrictions or serious infectious diseases. The anti-inflammatory and cell-proliferation-promoting activities of sPG are supported by studies using CS derived from other sources. The physiological significance of the molecular weight and sulfation pattern of sPG can be better understood by comparing it with those of CS derived from other raw materials. However, the precise mechanisms of action of sPG and other CS/CSPGs, particularly after oral administration, remain unknown. No absorption or translocation pathways were identified. Due to the poor absorption of CS/CSPG and the uncertainty of its mechanism, the conventional narrative that CS/CSPG is absorbed from the intestinal tract and reaches the affected area may not be sufficient. However, studies on the role of CS/CSPG in the intestinal tract are lacking. The possibility of prebiotic or luminacoid behavior in CS/CSPG has not yet received much attention. Their elucidation may shed light on the mechanisms of the oral administration of CS/CSPG and its benefits.

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