2021 Volume 27 Issue 3 Pages 453-462
β-Linked water soluble polysaccharides are important bioactive components of mushrooms and plants, they possess various biological activities, such as anti-inflammation, immunomodulation, anti-tumor and others. This study aimed to examine the comparative anti-inflammatory effects of five different β-linked water soluble polysaccharides from fungi and plants [i.e. Xylaria nigripes (XN), Grifola frondosa (GF), Lentinula lentodes (Len), Laminaria digitata (Lam) and Hordeum vulgare (BG)] in lipopolysaccharides-stimulated RAW264.7 macrophages. Although the selected five polysaccharides showed different potencies in anti-inflammatory activity, XN exhibited the strongest inhibitory effects on NO, TNF-α and IL-6 production, and iNOS and COX-2 expression, whereas the inhibitory activity of BG was the weakest. Among the polysaccharides with β-(1→3, 1→6) glucose linkages and triple-helix structures, the inhibition of GF and Len on TNF-α and IL-6 production was weaker than XN and Lam. This study concludes that the monosaccharide composition, glycosidic linkage and tertiary conformation were the main factors affecting the anti-inflammatory activity of polysaccharides, and polysaccharides with β-(1→3, 1→6) glycosidic linkages possessed stronger anti-inflammatory activity than β-(1→3, 1→4)-linked polysaccharides.
Pro-inflammatory mediators including nitric oxide (NO), interleukins-6 (IL-6), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are known to play diverse roles in inflammation and immune responses (Chen et al., 2018a). Dysregulation of these pro-inflammatory mediators is linked to various chronic diseases, such as cancer, rheumatoid artritis, cardiovascular diseases, Parkinson's and Alzheimer's diseases and others (Pawelec et al., 2014; Zhong and Shi, 2019). Upon responding to stimulation by foreign substances or pathogens, macrophages release pro-inflammatory mediators to drive more immune cells to the site of infection to protect against damage caused by pathogens (Arango Duque and Descoteaux, 2014; Liu et al., 2014). Thus, the inhibition of proinflammatory mediators released by activated macrophages has been considered to be a useful strategy for evaluating natural products in the prevention of inflammation-associated diseases (Liu et al., 2014).
β-Linked water soluble polysaccharides including β-glucan are important bioactive constituents of mushrooms and plants; they are large polymeric molecules composing of monosaccharide molecules linked by glycosidic bonds, and are resistant to digestion in the small intestine. Studies have demonstrated that these water soluble polysaccharides possessed various biological activities including anti-inflammation, immunomodulation, anti-tumor, hypoglycemia and hypolipidemia (Zhang et al., 2007; Giavasis, 2014; Du et al., 2015), and their potencies are affected by the primary structure, molecular weight, monosaccharide composition, linkage type, degree of branching, solubility, protein content, conformation and others (Lam and Cheung, 2013; Su et al., 2016).
Polysaccharides from Xylaria nigripes, Grifola frondosa, Lentinula lentodes, Laminaria digitata (brown algae) and Hordeum vulgare (Barley) have been used for treating and preventing chronic diseases. X. nigripes, G. frondosa and L. lentodes are popular edible medicinal mushrooms in Asia, whereas L. digitata and H. vulgare are photosynthetic organisms. The molecular weight of X. nigripes water soluble polysaccharides (XN) was reported to be about 900 kDa, and composed of mainly glucose (ca. 90%), and has a triple-helix configuration (Chen et al., 2018b). The water soluble polysaccharides of G. frondosa (GF) contained primary and secondary populations with the molecular weights of 891.1 kDa and 21.2 kDa, respectively; it is a heterogeneous polysaccharide with triple-helix structure, and is made up of fucose, galactose, glucose, mannose and ribose (Su et al., 2017). Lentinan (Len), an important water soluble polysaccharide of L. lentodes with molecular weight of about 500 kDa; it is a homogeneous polysaccharide composed of mainly glucose, and possesses triple-helix conformation (Zong et al., 2012). Laminarin (Lam), a water soluble triple-helical polysaccharide of L. digitata, is a low molecular weight polysaccharide (2–3 kDa) comprised mainly glucose (Graiff et al., 2016). Barley β-glucan (BG) is a water soluble homopolysaccharide with molecular weight of 31–2700 kDa, its glucose monomers are linked via β-(1→3,1→4) glycosidic bonds and mostly exists in an irregular spiral form (Lazaridou et al., 2007). Unlike BG [β-(1→3,1→4)], XN, GF, Len and Lam are mainly made up of monosaccharides that are linked by β-(1→3,1→6) glycosidic bonds.
Studies have shown that XN possessed hepatoprotective, antioxidant (Song et al., 2011), and anti-inflammatory (Ko et al., 2011) activities. GF was reported to have anti-inflammatory activity (Meng et al., 2016; Su et al., 2017); its D-fraction is commercially used for treating cancer and HIV (Mayell, 2001). Len was shown to have good anti-inflammatory (Xu et al., 2012; Nishitani et al., 2013), anti-cancer and immunomodulatory (Zong et al., 2012; Wang et al., 2020) activities. Lam has many reported biofunctional activities including anti-tumor, anti-inflammatory, anti-coagulant and antioxidant activities (Kadam et al., 2015). BG has been demonstrated to possess anti-inflammatory (Arcidiacono et al., 2019), hypocholesteremic and blood glucose lowering effects (Tiwari and Cummins, 2011).
Although the health benefits of mushroom and plant polysaccharides have received increasing attention in the scientific communities and the consumers, current scientific understanding of this class of ingredients is largely specific to an individual type of mushroom or plant. There are very few systematic comparative studies on the anti-inflammatory activity of different sources of polysaccharides that have different physicochemical properties. In this study, the anti-inflammatory activity of five selected water soluble polysaccharides, namely XN, GF, Len, Lam and BG from different sources with different physicochemical properties were compared for their effects on pro-inflammatory mediator production in LPS-induced RAW264.7 macrophage cells.
Chemicals Laminarin (Lam), barley β-glucan (BG), bovine serum albumin (BSA), lipopolysaccharides (LPS), α-amylase (Aspergillus oryzae, EC 3.2.1.1) and α-glucosidase (Saccharomyces cerevisiae, EC 3.2.1.20), anti-iNOS antibody, anti-COX-2 antibody, anti-β-actin antibody, anti-mouse IgG antibody and anti-rabbit IgG antibody were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Lentinan (Len) was obtained from Shanghai Biochempartner Co., Ltd. (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco (Grand Island, NY, USA). All other chemicals used were of analytical grade.
Preparation of β-linked water soluble polysaccharides of X. nigripes and G. frondosa Previous studies have shown that X nigripes (XN) and G. frondosa (GF) water soluble polysaccharides possess good anti-inflammatory activity (Su et al., 2017; Chen et al., 2018b), hence the same purified polysaccharides were prepared and used in this study. In brief, both XN and GF were prepared from dried fruiting bodies of mushrooms that were provided by Kang Jian Biotech Co., Ltd. (Nantou, Taiwan), they were extracted and purified according to procedures described previously (Su et al., 2017; Chen et al., 2018b).
Cell culture RAW264.7 (BCRC No. 60001), a murine macrophage cell line, was obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified 5% CO2 atmosphere.
Cell viability assay The effects of test samples on cell viability were evaluated by MTT assay. In brief, RAW264.7 macrophages were seeded in a 48-well culture plate at a density of 1.2 × 105 cells per well, and then incubated for 24 h. Cells were pre-treated with 300 µL of various concentrations of samples (50, 100 or 200 µg/mL) at 37 °C for 1 h and then stimulated with LPS (1 µg/mL) for 24 h. The culture supernatant was collected and 100 µL of culture medium containing 2.5 mg/mL MTT was added into each well. After incubation at 37 °C for 4 h, the supernatant was discarded and the formazan crystal formed in the cells was dissolved with 300 µL of dimethyl sulfoxide. The absorbance at 570 nm was measured by a microplate reader (Infinite M200 PRO, Tecan, Morrisville, NC, USA).
Measurement of nitric oxide The nitric oxide (NO) production was determined by measuring the nitrite concentration in the culture media. RAW264.7 macrophages (5 × 104 cells per well) were seeded in 96-well culture plates and incubated for 24 h before treatment. Cells were treated with or without samples (XN, GF, Len, Lam and BG) at various concentrations (50, 100 or 200 µg/mL) at 37 °C for 1 h, followed by stimulation with LPS (1 µg/mL) for 24 h. The supernatant (100 µL) from each well was mixed with 100 µL Griess reagent [containing 1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride and 2.5% phosphoric acid]. After 10 min of incubation at room temperature, the absorbance was measured with a microplate reader (Infinite M200 PRO, Tecan) at 540 nm. The nitric oxide concentration was calculated from a sodium nitrite calibration curve.
TNF-α and IL-6 production assays RAW264.7 macrophages were seeded in a 96-well culture plate (5 × 104 cells per well) and incubated for 24 h before treatment. Cells were treated with samples at various concentrations (50, 100 and 200 µg/mL) at 37 °C for 1 h, followed by stimulation with LPS (1 µg/mL) for 24 h. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured using ELISA kits according to the manufacturer's instructions (BD Biosciences, San Diego, CA, USA).
Western blot analysis of iNOS and COX-2 expression RAW264.7 macrophage cells (4 × 105 cells per well) were seeded in a 6-well plate for 24 h at 37 °C, followed by treating with various concentrations of samples for 1 h, and then treated with LPS (1 µg/mL) for 24 h. After washing the cells twice with cold phosphate-buffered saline (PBS), the cell suspension was collected in a centrifuge tube and centrifuged at 500 g at 4 °C for 5 min. After removing the supernatant, 100 µL SubCell Buffer I (containing 1x Protease Arrest, G-Biosciences) was added and shaked vigorously for 15 s, followed by placing on ice for 10 min. After adding 5 µL of SubCell Lysis Reagent and shaked vigorously for 5 s, and then placed on ice for 1 min, cell debris was removed by centrifuging at 16 000 g at 4 °C for 5 min. The supernatant was collected for iNOS and COX-2 expression analysis, and its protein content was determined according to the method described by Bradford (1976).
Fifty micrograms of cellular protein from treated and untreated cell extracts was electroblotted onto a polyvinylidene difluoride (PVDF) membrane following separation on a 10% SDS-polyacrylamide gel electrophoresis. The immunoblot was incubated overnight with blocking solution (5% skim milk) at 4 °C, followed by incubation for 4 h with a primary antibody (iNOS and COX-2 antibodies at 1:5 000 dilution, and β-actin antibody at 1:8 000 dilution). Blots were washed four times with Tween 20/Tris-buffered saline (TTBS) and incubated with a 1:5 000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were again washed three times with TTBS and then developed by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). The MutiGel-21 cold light imaging system was used for development and analysis, and ImageJ software was used for quantification.
Statistical analysis All values are expressed as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) and post-hoc Tukey-Kramer tests were applied to assess the statistical significance of the differences among means of the study groups (SPSS version 10.0, Chicago, USA). A value of p < 0.05 was considered as statistical significance.
Yields of crude polysaccharides and β-linked water soluble polysaccharides of X. nigripes and G. frondosa Results showed that the yields of crude polysaccharides obtained from fruiting bodies of X. nigripes and G. frondosa were 4.32% and 7.66%, respectively (Table 1). After further purification, the yields of β-linked water soluble polysaccharides (XN and GF) were 2.39% and 4.18%, respectively (Table 1), of which the yields were about half of that of their respective crude polysaccharides, indicating that the crude polysaccharides account for 50% of non-digestible polysaccharides; these results are consistent with results reported by Chen et al. (2018b) and Su et al. (2017). In this study, digestible polysaccharides such as starch (α-glucan) were broken down by α-amylase and α-glucosidase, and they were removed from the samples during dialysis. A small amount of protein was found in both XN and GF, they were 1.01% and 0.49%, respectively. These proteins are probably bound to the soluble glucans, and hence the Sevag method was not able to remove them completely.
| Sample | Yield (%, DW) | |
|---|---|---|
| Crude polysaccharides | β-Linked water soluble polysaccharides | |
| X. nigripes | 4.32 ± 0.75 | 2.39 ± 0.37 |
| G. frondosa | 7.66 ± 0.40 | 4.18 ± 0.26 |
Data are presented as mean ± SD (n = 3); DW: dry weight.
Cell viability In order to ensure the selected concentrations of XN, GF, Lam, Len, BG and LPS for evaluation of anti-inflammatory activity were not toxic to RAW264.7 macrophage cells, cell viability assessment was performed with MTT assay. Results showed that at concentrations 0, 50, 100 and 200 µg/mL of test samples plus 1 µg/mL LPS demonstrated no cytotoxic effect on RAW264.7 cells (Fig. 1). All treatments and the control group showed a survival rate of above 95%, indicating that the LPS and polysaccharide samples cause no toxicity to cells within the tested concentration range. Therefore, this concentration range was selected for subsequent anti-inflammatory experiments.
Effects of different treatments on the viability of LPS-stimulated RAW264.7 macrophages. (a) XN, (b) GF, (c) Lam, (d) Len and (e) BG. The cells were pretreated with different concentrations of samples (50, 100 and 200 µg/mL) for 1 h and then treated with or without LPS (1 µg/mL) for 24 h. Data are presented as mean ± SD (n = 4). The bars having different letters are significantly different (p < 0.05) as analyzed by Tukey-Kramer tests.
Inhibition on nitric oxide production Nitric oxide (NO) is an inflammatory mediator that modulates immune responses. Upon stimulation by LPS, the activated macrophages secrete a large amount of NO, which promotes blood vessel expansion and trigger immune responses to resist the invasion of foreign substances. However, when NO is over accumulated in cells or tissues for a long period of time, it may cause chronic inflammation and resulted in chronic diseases such as cancer, diabetes, hypertension, arthritis and others (Nussler and Billiar, 1993; Liaudet et al., 2000). Therefore, the amount of NO produced has been used as an important index to indicate the degree of inflammation.
Figure 2a shows the different effects of the selected five polysaccharides on NO production in LPS-stimulated RAW264.7 macrophages. Results showed that LPS caused significantly increased in NO production, however the NO production was significantly reduced with good dose-dependent effects after XN, GF, Lam and Len treatments; among them, XN appears to have the strongest inhibitory activity on NO production. At 200 µg/mL, XN, GF, Len and Lam showed good NO production inhibitory effect, and their inhibition rates were 62.9%, 59.3%, 54.7% and 44.2%, respectively; however, the inhibitory effect of BG was minimal, with only 9.0% inhibitory effects on NO production in LPS-induced RAW264.7 macrphages. These results indicate that XN, GF, Len and Lam with β-(1→3, 1→6) linkages and triple-helix configurations have a stronger inhibitory activity on NO production than BG that has a mixture of β-(1→3, 1→4) glycosidic bonding and without triple-helical structure. This finding was consistent with the observation of Su et al. (2017), pointing out that the content of β-(1→3, 1→6) glucan is a factor that affects the production of NO. Enshasy and Hatti-Kaul (2013), and Yanaki et al. (1986) have also pointed out that polysaccharides with triple-helix conformation were more stable and had stronger bioactivities.
Effects of different concentrations of polysaccharides (XN, GF, Lam, Len and BG) on nitric oxide (NO), TNF-α and IL-6 production in LPS-stimulated RAW264.7 macrophages. RAW 264.7 macrophage cells were pretreated with different concentrations of samples (50, 100 and 200 µg/mL) for 1 h and then treated with or without LPS (1 µg/mL) for 24 h. (a) NO, (b) TNF-α and (c) IL-6. Data are presented as mean ± SD (n = 4). The bars having different letters are significantly different (p < 0.05) as analyzed by Tukey-Kramer tests.
Inhibition on TNF-α and IL-6 secretions In addition to play the role of molecular messengers between immune cells, cytokines also have the functions in activating T cells and white blood cells, as well as regulating growth and differentiation, etc. In general, when stimulated by foreign substances or pathogens, cells secrete cytokines to transmit messages to activate immune responses. For example, the LPS-stimulated macrophages release cytokines such as TNF-α and IL-6, resulting in the migration of immune cells to the site of infection and injury, and consequently triggering an inflammatory response in the body to resist the invasion of foreign substances (Arango Duque and Descoteaux, 2014). Therefore, TNF-α and IL-6 are regarded as pro-inflammatory cytokines, which are commonly used to assess the degree of inflammation.
Figure 2b shows the results of LPS-induced TNF-α secretion by different concentrations of XN, GF, Len, Lam and BG. Results showed that LPS significantly increased TNF-α secretion in RAW264.7 macrophages, however, the addition of XN, GF, Len, Lam and BG significantly reduced the secretion of TNF-α in a dose-dependent manner. At 200 µg/mL, XN and Lam had the best inhibitory effect on TNF-α secretion, with an inhibition rate of 41.9% and 37.5%, respectively. Although Len and GF also showed inhibitory effects on TNF-α secretion, their inhibition rates were only about half of that of XN and Lam, with inhibition rates 23.4% and 18.4% respectively. The reason may be that XN and Lam are both homopolysaccharides that are composed of mainly glucose molecules; this indicates that the immunomodulatory activity of homopolysaccharides could be better than that of heteropolysaccharides. For mixed linear β-(1→3, 1→4)-linked glucans, BG has the weakest inhibitory effect on TNF-α secretion. Previous studies have also pointed out that the mixed linear β-(1→3, 1→4) glucan showed weaker immunomodulatory activity than that of β-(1→3)-bonded glucan (Demleitner et al., 1992), and the cytokine inducing activity decreased when triple-helix structures of the polysaccharides were destroyed (Falch et al., 2000). Therefore, the present results suggest that polysaccharides with β-(1→3, 1→6) linkage, triple-helix structure and monosaccharides composed of glucose molecules may play an important role in inhibiting TNF-α production in LPS-induced RAW264.7 macrophages.
IL-6 was significantly increased in LPS-stimulated RAW264.7 macrophages, but it was significantly reduced by XN, Lam, Len and GF treatments (Fig. 2c). At 200 µg/mL, XN and Lam had the strongest inhibitory effect on IL-6 secretion, with inhibition rates of 50.1% and 42.7%, respectively, followed by Len and GF, while BG exhibited no significant inhibition on IL-6 production. These results also indicate that polysaccharides with β-(1→3, 1→6) glycosidic binding and triple-helix configuration have better inhibitory effect on IL-6 secretion in LPS-induced RAW264.7 macrophages than BG, which has linear β-(1→3, 1→4) linkaging bonds.
Based on the above observations, it was noted that although all β-(1→3, 1→6)-linked polysaccharides with triple-helix structures have inhibitory effects on TNF-α and IL-6 secretions in LPS-induced RAW264.7 macrophages, the homopolysaccharides containing mainly glucose units have better TNF-α and IL-6 secretion inhibitory rates than those of heteropolysaccharides with β-(1→3, 1→6) glycosidic linkages, while the inhibitory effect of mixed linear β-(1→3, 1→4) polysaccharides is less effective. Previous studies have shown that the production of NO has positive co-relation with the secretion of other pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β (Bonizzi et al., 2004; Dawn et al., 2004), indicating that XN, GF, Len and Lam may also indirectly reduce NO secretion by inhibiting pro-inflammatory cytokine production.
Inhibition of iNOS and COX-2 expression Both iNOS and COX-2 proteins are well known to play an important role in inflammation. iNOS converts L-arginine to NO, which is a gaseous molecule that affects vasodilation, platelet function and promotes inflammation; whereas COX-2 converts arachidonic acid to PGE2, which regulates platelets agglutination, renal bleeding and inflammation. Excessive production of NO and PGE2 may cause abnormal inflammation and lead to increasing risk of chronic diseases such as cancer, arthritis, diabetes and others (Park et al., 2003; Chen et al., 2018a). Therefore, regulating the expression of upstream proteins (such as iNOS and COX-2) may reduce the output of downstream pro-inflammatory mediators, thereby achieving the suppression on the occurance of chronic inflammation.
Figures 3a and 3b show the effect of different concentrations of XN, GF, Lam, Len and BG on the iNOS expression. The results showed that LPS significantly upregulated the expression of iNOS, however it was significantly down-regulated by XN, GF, Lam and Len treatments, and the suppression was significantly enhanced with increasing dose of treatments. Although the BG treatment showed inhibitory effect on iNOS expression, its inhibition rate at concentration of 200 µg/mL was only half of that of other polysaccharides (Fig. 3b). XN demonstrated the strongest inhibitory activity on iNOS expression, with an inhibition rate of 61.5%, followed by Len (50.1%), GF (43.3%) and Lam (32.4%). The inhibitory effect of BG on iNOS expression at 200 µg/mL was only 16.6%, indicating that the inhibitory effect of BG on iNOS expression was much weaker than the other four polysaccharides. Previous studies have found that β-(1→3, 1→6) glucan in black yeast (Aureobasidium pullulans) can effectively inhibit LPS-induced iNOS expression, thereby reducing NO production (Choi et al., 2016), while BG with the mixed linear β-(1→3, 1→4) linkage has poorer anti-inflammatory activity than β-(1→3)-linked glucans. In this study, XN, Len, GF, and Lam with β-(1→3, 1→6) linkages and triple-helix configuration have good inhibitory effects on the expression of iNOS, and their inhibitory potency on iNOS expression was in line with the trend of NO production.
Effects of different concentrations of polysaccharides (XN, GF, Lam, Len and BG) on iNOS and COX-2 expression in LPS-stimulated RAW264.7 macrophages. Cells were pretreated with different concentrations of samples (50, 100 and 200 µg/mL) for 1 h and then treated with or without LPS (1 µg/mL) for 24 h. β-actin was used as a loading control. (a) and (c) A representative photograph of Western blot; (b) and (d) Western blotting quantitative data was estimated relative to the loading control protein from each analysis of different samples. Data are presented as mean ± SD (n = 3). The bars having different letters are significantly different (p < 0.05) as analyzed by Tukey-Kramer tests.
Figures 3c and 3d show the differences in the expression of COX-2 protein at different concentrations of XN, GF, Lam, Len and BG. The results showed that the COX-2 expression was not obvious in the group without treatments (Con). However, the expression of COX-2 was significantly increased in the presence of 1 µg/mL LPS, while the expression level was significantly down-regulated by the treatment of XN, GF, Lam, Len and BG. At concentration 200 µg/mL, Len, XN and Lam exhibited the strongest inhibitory effect on COX-2 expression, with the inhibition rates of 43.6%, 38.6% and 30.0%, respectively. Previous studies have pointed out that pro-inflammatory cytokines TNF-α and IL-1β positively regulate the expression of COX-2 protein (Medeiros et al., 2010; Szymanski et al., 2012). In this study, the trend of inhibition on TNF-α secretion by XN, GF, Lam, Len and BG was similar to COX-2 expression, indicating that the inhitory effect on COX-2 expression by these polysaccharides may be affected by TNF-α. Similarly, XN, Len, GF and Lam with β-(1→3, 1→6) linkages and triple-helix conformation exhibited stronger inhibitory effects on iNOS and COX-2 expression than no triple-helical structure β-(1→3, 1→4)-D-glucan of barley (BG).
Although Dectin-1 (a β-glucan receptor) is believed to be highly specific for glucans with a pure (1→3)-β-linked backbone structure, however it does not bind to all (1→3)-β-linked glucans with similar affinity (Adams et al., 2008). Structural analysis demonstrated that glucan backbone chain length and (1→6)-β-side-chain branching strongly influenced Dectin-1 binding affinity. When β-glucan binds to Dectin-1, it initiates a series of immune responses, including promoting the production of phagocytosis and regulating the secretion of pro-inflammatory mediators (Goodridge et al., 2009). Nevertheless, the present results show that polysaccharides containing β-(1→3, 1 →6) glucans exhibited stronger inhibitory effects on pro-inflammatory mediator production than polysaccharides with β-(1→3, 1→4) linkages in LPS-stimulated RAW264.7 macrophages, suggesting the involvement of other immune receptors (such as TLR-2, TLR-4 and CR3) in the regulation of immune responses (Chen and Seviour, 2007; Su et al., 2020).
The degree of branching of β-(1→3, 1→6) glucans in XN, GF, Lam and Len were 0.09, 0.36, 0.14 and 0.40 respectively (Hrmova and Fincher, 1993; Enshasy and Hatti-Kaul, 2013; Su et al., 2017), whereas BG was a linear β-glucan, and it has zero degree of branching (DB = 0). It has been reported that active polysaccharides generally have a degree of branching between 0.20 and 0.33 (Enshasy and Hatti-Kaul, 2013), however our results showed that XN (0.09) and Lam (0.14), which had lower degree of branching than GF (0.36) and Len (0.40), but had stronger anti-inflammatory activities, and the nonbranching BG had the worst anti-inflammatory activity. These results further suggest that besides the degree of branching, types of linkage and tertiary structure, other physicochemical factors may have contributed to the differences in anti-inflammatory activity between XN, GF, Lam and Len.
Under LPS-stimulated RAW264.7 macrophage cell model of inflammation, it was found that the five β-linked water soluble polysaccharides (XN, GF, Lam, Len and BG) possessed a varying degree of inhibitory activity on pro-inflammatory mediator production within the concentration range that causes no cell death toxicity. Although these five polysaccharides possessed inhibitory effect on the production of NO, IL-6 and TNF-α, and the expression of iNOS and COX-2 in LPS-stimulated RAW264.7 macrophages, XN and Lam exhibited the best inhibitory activity while the activity of BG was the weakest. The present study concludes that monosaccharide composition, glycosidic bond type and tertiary conformation may be the main factors contributing to the differences in anti-inflammatory activity of β-linked water soluble polysaccharides from the selected fungi and plants, and the triple-helical polysaccharides with β-(1→3, 1→6)-linked glucose units exhibited stronger anti-inflammatory activity than the polysaccharides with β-(1→3, 1→4) glycosidic linkages and irregular spiral structures.
Acknowledgements The authors would like to thank the Ministry of Science and Technology of Taiwan for partial funding of this study under grant number MOST 104-2622-B-002-006-CC2.
Conflict of interest The authors declare no conflicts of interest.
