Arabinogalactans from Larix principis-rupprechtii: An investigation into the structure-function contribution of side-chain structures

Carbohydrate Polymers 227 (2020) 115354

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Arabinogalactans from Larix principis-rupprechtii: An investigation into the structure-function contribution of side-chain structures

T

Shuo Tanga,b,c, Ting Wanga,b,c, Caoxing Huanga,b,c, Chenhuan Laia,b,c, Yimin Fana,b,c, ⁎ Qiang Yonga,b,c, a

Key Laboratory of Forestry Genetics & Biotechnology (Nanjing Forestry University), Ministry of Education, Nanjing 210037, People’s Republic of China Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China c Jiangsu Province Key Laboratory of Green Biomass-based Fuels and Chemicals, Nanjing 210037, People’s Republic of China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Larix principis-rupprechtii Arabinogalactan Side chains Smith degradation Immunomodulatory

Certain polysaccharides can serve as bio-active natural polymers that provide health benefits, however their complex structures tend to induce dramatically different activities. In this work, the immunomodulatory activity of the two arabinogalactans from larch was investigated, based upon comparison of native arabinogalactans and those modified to have lesser quantities of side-chain paraphernalia. Various in vitro assays demonstrated that the native arabinogalactans increased the secretion of macrophage-derived biological factors including NO, TNFα, IL-1β and IL-6. Methylation analysis and nuclear magnetic resonance spectra results indicated that part side chains of arabinogalactan were successfully removed. Partial removal of the side-chains enhanced immunomodulatory activity, while excessive removal resulted in a sharp decrease in immunomodulatory activity. These results provide new evidence that alludes to the structure-function relationship of bio-active polysaccharides, furthering the case for development of a universal assessment of polysaccharide structure-function proclivities.

1. Introduction The genus Larix (larch) is a softwood plant that can be found widely distributed across regions of China, Russia, Canada, Central Europe, and other cool, temperate regions of the northern hemisphere. Larix principis-rupprechtii is one varieties of larch that is specifically pervasive across northeastern China (Mason & Zhu, 2014). A characteristic feature of larch is the presence of arabinogalactans (AGs) within the wood, which can be extracted from larch trees at high yields. In the mid-20th century, a number of researchers have begun to study the structure and function of AG from western larch (Churms, Merrifield, & Stephen, 1978; Kelly, 1999). Research showed that AG is a water-soluble polysaccharide that features a high degree of intramolecular branching. In general, AGs are constituted by a (1→3)-β-D-galactopyranosidic backbone that branches at position 6 to mono- or oligosaccharide side chains consisting of Galp, Araf, Arap and GlcpA (Goellner, Utermoehlen, Kramer, & Classen, 2011; Tang et al., 2018). Larch AGs are approved by the Food and Drug Administration (FDA) of USA as a source of dietary

fiber, but reports suggest that there are additional therapeutic benefits to consumption of AGs such as immune stimulation (Kelly, 1999). Larch AG’s effects on the immune system have been investigated through multiple human studies with different objectives (Riede, Grube, & Gruenwald, 2013; Robinson, Feirtag, & Slavin, 2001; Udani, Singh, Barrett, & Singh, 2010). Across these studies, each concluded that larch AG provided benefits to human health in the form of a variety of positive effects. Generally, polysaccharides are considered to be biological response modifiers based upon their antitumor and immuno-modulating activities. These biological activities have been found to be associated with monosaccharide composition, molecular weight, types of glycosidic linkages, degree of branching, and specific tertiary conformations (Bohn & Bemiller, 1995; Sletmoen & Stokke, 2008). To date, the structure and function of numerous forms of polysaccharides from different sources have been extensively studied (Ferreira, Passos, Madureira, Vilanova, & Coimbra, 2015). However, an exact understanding of structure–function relationships across polysaccharides

⁎

Corresponding author at: Key Laboratory of Forestry Genetics & Biotechnology (Nanjing Forestry University), Ministry of Education, Nanjing 210037, People’s Republic of China. E-mail addresses: [email protected] (S. Tang), [email protected] (T. Wang), [email protected] (C. Huang), [email protected] (C. Lai), [email protected] (Y. Fan), [email protected] (Q. Yong). https://doi.org/10.1016/j.carbpol.2019.115354 Received 25 July 2019; Received in revised form 15 September 2019; Accepted 19 September 2019 Available online 21 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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2.2. Smith degradation

remains unattained. Evidence suggests that the activity of polysaccharides is dependent on their size, with medium molecular weight (5–20 kDa) fractions exhibiting more potent immunostimulant properties (Stephanie, Eric, Sophie, Christian, & Yu, 2010). It has been noted that there is a positive relationship between the degree of branching in a given polysaccharide and its ensuing immunological activity, something that was also related to the bond type of the branching structures (Lee et al., 2010; Mueller et al., 2000; Togola et al., 2008). Regarding molecular confirmations and immunostimulation, studies have shown that triple-helix and random coil conformation confer higher immunostimulatory activity to polysaccharides (Lee et al., 2010; Satitmanwiwat et al., 2012; Yin et al., 2012). In spite of the well-established knowledge, it is still necessary to conduct a targeted study of the exact structural and functional relationships for polysaccharide through chemical or biological means. One avenue for polysaccharide biological investigation is through use of immunological model of macrophages. Macrophages play an important anti-inflammatory role and regulate immune response through the release of cytokines such as tumor necrosis factors (TNF), interleukins (IL), and interferons (IFN) (Mills, 2012). Immunostimulatory activities of polysaccharides may be due to direct or indirect interactions with these immune system components, resulting in the triggering of diverse cellular and molecular events (Leung, Liu, Koon, & Fung, 2006). Reportedly, polysaccharides are involved in the stimulation of various cytokines and chemokines by activating receptors on the cell membrane to activate mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, thereby exerting immune-potentiating effects (Yan et al., 2018; Zhang et al., 2013). The current experiments were designed to investigate the immunomodulatory activity on the murine macrophage cell line, RAW 264.7 cells, by determining the production of NO, TNF-α, IL-1β, and IL6. In our previous study, we successfully isolated and purified two arabinogalactan AGW (Elution from AGs by DEAE-Sepharose with water) and AGS (Elution from AGs by DEAE-Sepharose with salt). AGW and AGS have a 1,3-linked Galp backbone, branched at C6 to 1,6-linked Galp side residues terminated by Galp, Arap, Araf or GlcpA, wherein the AGS contains higher uronic acid and greater molecular weight (Tang et al., 2018). After establishing this reproducible method, in this paper, we focus on the immunomodulatory activity of AGW and AGS on macrophage cells in effort to promote further valorization of these sustainable bioactive compounds. Moreover, we also experimented with the effects of modifying AGW by way of side-chain removal to better probe for specific structure-function relationships in the tested biological system. In all, this work aims to further the state of knowledge concerning the side-chain trimming effect on the in vitro immunomodulatory activity for larch-derived AG while simultaneously demonstrating their value as a biologically active.

Smith degradation was carried out according to a previous method with minor modifications (Goldstein, Hay, Lewis, & Smith, 1965). Briefly, 10 g of AGW was oxidized in 1000 ml of NaIO4 (0.1 M) under darkness for 5 days at 4 °C. The reaction was stopped by addition of 5 ml of ethylene glycol, and next neutralized to pH 7.0 using 5 M NaOH. The solution was concentrated by rotary evaporation to a volume of 200 ml and then dialyzed against water for 3 days at 4 °C. The dialyzed product was next reduced with 5 g of sodium borohydride overnight in the dark place. Following borohydride reduction, the product was neutralized to pH 7.0 with acetic acid, and then dialyzed against water for 2 days. Trifluoroacetic acid (TFA) was added to a final concentration of 1 M, after which the product was hydrolyzed at room temperature for 12 h. The insoluble compounds produced were then removed by centrifugation at 9600 g for 10 min. The aqueous fraction was next precipitated with three volumes of 95% ethanol and kept at 4 °C overnight. The precipitate was freeze-dried to obtain degraded AGs, termed DAG1 (Once Degraded Arabinogalactans). To produce a product containing even further side chain removal, the entire process was repeated upon DAG1 to yield a new product referred to as DAG2 (Twice Degraded Arabinogalactans). 2.3. Monosaccharide composition analysis of modified arabinogalactans The monosaccharides composition of DAG1 and DAG2 was determined by high performance anion-exchange chromatography (HPAEC) according to the method described by (Tang et al., 2018). Briefly, ˜10 mg the samples were hydrolyzed with 2 M TFA at 121 °C for 1 h. After hydrolysis, the solvent was then removed by rotary evaporation at 30 °C. The residue was washed with methanol several times to remove excess TFA. Resultant monosaccharides in hydrolysate were analyzed by a HPAEC system (Dionex ICS-5000, USA) equipped with a CarboPac™ PA10 column (2 × 250 mm) and a pulsed amperometric detector. The elution program consisted of an initial isocratic elution in 37 mM NaOH from 0 to 20 min, followed 200 mM CH3COONa from 20 to 35 min, and finally equilibrated in 37 mM NaOH from 35 to 50 min. Sugar identification involved direct comparison with commercial monosaccharide standards. 2.4. FT-IR spectroscopy Fourier-transform infrared spectra (FT-IR) were recorded over the wavenumber range of 4000–400 cm−1, using a VERTEX 80 V (Bruker, Germany) spectrometer with the KBr-disk method. 2.5. Methylation analysis

2. Materials and methods

Methylation of DAG1 and DAG2 was conducted according to the method of previous report with some modification (Hakomori, 1964). Briefly, polysaccharide samples (10 mg) were dissolved in 5 mL of DMSO until clarified. Subsequently, 200 mg of NaH was added to the solution, then mixed with 2 mL of methyl iodide and kept in darkness for 12 h at 25 °C. After time, the solution was extracted four times with chloroform. The chloroform layer was then concentrated and dried. Methylated products were next hydrolyzed with 2 M TFA at 105 °C for 6 h. The hydrolysate was reduced with NaBH4 (10 mg) for 4 h at room temperature. Reduction was then followed by an acetylation step utilizing acetic anhydride (0.5 mL) and pyridine (0.5 mL) for 2 h at 100 °C. Methylated alditol acetates were finally analyzed by a Thermo Trace ISQ GC–MS system (Thermo Fisher Scientific, USA). The initial column temperature was set at 80 °C (held for 2 min), and programmed to 280 °C (held for 10 min) at 10 °C/min. The partially methylated alditol acetates were identified by their mass spectra and relative retention time.

2.1. Materials and reagents Arabinogalactans (AGW and AGS) were isolated and purified from Larix principis-rupprechtii according to our previous report (Tang et al., 2018). Deuterium oxide (D2O) and monosaccharide standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Hyclone (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) and Nitric Oxide (NO) assay kits were obtained from Beyotime (Shanghai, China). Enzyme linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β and IL-6 were all purchased from Boster Biological Technology (Wuhan, Hubei, China). MiniBEST Universal RNA Extraction Kit, PrimeScript RT reagent Kit with gDNA Eraser and SYBR Premix Ex Taq II Kit were each acquired from TaKaRa Biotechnology (Dalian, Liaoning, China). All other employed reagents were analytical grade. 2

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5′-CGGCAAACATGACTTCAGGC-3′; Reverse: 5′-GCACATCAAAGCGGC CATAG-3′), TNF-α (Forward: 5′-AGCCCCCAGTCTGTATCCTT-3′; Reverse:5′-TGATGGTGGTGCATGAGAGG-3′), IL-1β (Forward:5′-TTCA GGCAGGCAGTATCACTC-3′; Reverse: 5′-GAAGGTCCACGGGAAAGA CAC-3′), IL-6 (Forward: 5′-TCTACTCGGCAAACCT-3′; Reverse: 5′-ATA GTGTCCCAACATTCA-3′).

2.6. NMR analysis Each polysaccharide (40 mg) was dissolved in deuterium oxide (D2O, 0.5 mL) at room temperature. DSS (4,4-dimethyl-4-silapentane-1sulfonic acid) was used as standard. 13C spectra were recorded on a Bruker AVANCE 600 MHz spectrometer (Bremen, Germany) using standard Bruker pulse sequences at 25 °C. 13C chemical shifts were acquired in relation to DSS, which was calibrated externally.

2.9. Statistical analysis

2.7. Congo red test

All experiments were carried out at least in triplicate, and results were expressed as mean ± SD. Statistical analysis was performed using a student's t-test and SPSS 20 software. Difference with P < 0.05 was considered statistically significant.

Conformational structures of the polysaccharides were determined by Congo-red test. Briefly, a polysaccharide solution (2 mL, 2 mg/mL) was mixed with 2 mL Congo red solution (0.2 mM) and 1 mL various concentrations of NaOH, to give a final NaOH concentration ranging between 0–1 M. After equilibrating for 10 min in darkness, the maximum absorption wavelength (λmax) was measured using UV–vis spectrophotometer (UV-1800, Shimadzu, Japan) in a 400–600 nm range.

3. Results and discussion 3.1. Monosaccharide composition In order to study the effect of side chain on the activity of polysaccharides, some of the side chain glycosyl groups were removed by smith degradation method. Finally, the yield of DAG1 and DAG2 were 42.31% and 25.46% based on the weight of AGW, respectively (Table 1). According to the results of monosaccharide composition and linkage analysis described in our previous report, the fundamental structure of AGW and AGS included a β-1,3-linked galactose backbone with β-1,6-linked galactose and arabinose side chains, and the ratios of galactose to arabinose were 9.92 and 11.14, respectively (Tang et al., 2018). In the present research, AGW was partially degraded by the Smith degradation method to remove side-chain structures in attempt to understand their effects on polysaccharide function. Monosaccharide analysis showed that both DAG1 and DAG2, degraded AG samples, consisted of 5.05% Ara and 94.95% Gal, or 2.16% Ara and 97.84% Gal, respectively (Table 1). The molar ratios of Ara to Gal for DAG1 and DAG2 were 1:18.8 and 1:45.3, respectively. These results indicated that Smith degradation caused part of the monosaccharide to detach from the original polysaccharide, which changed the proportion of monosaccharides with respect to their starting material. Smith degradation is able to cleave sugar residues containing vicinal hydroxyl groups, therefore only O-3-linked hexoses should survive this treatment. Meanwhile terminal sugars and hexoses in any other residues would be degraded (Kitazawa et al., 2013). Therefore, Gal and Ara in the side chain are continually degraded during Smith degradation, eventually leading to an increase in the ratio of Gal and Ara.

2.8. Immunomodulatory activity 2.8.1. Cell culture RAW 264.7 cells were purchased from Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS, China) and cultured in DMEM supplemented with penicillin (100 units/mL), streptomycin (100 units/mL) and 10% (v/v) fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. 2.8.2. Cell viability assay The effect of the polysaccharides on the viability of RAW264.7 cells was determined using CCK-8 kits and a method provided by the manufacturer. To begin, cells were loaded into the 96-well plate at a density of 1 × 105 cells/mL, and then kept for 24 h at 37 °C in a humidified incubator with 5% CO2 atmosphere, and then were cultured with different concentrations of samples for 24 h. Finally, 10 μL of CCK-8 regent was added to each well and further incubation took place over 1 h at 37 °C & 5% CO2. After incubation, absorbance at 450 nm was detected by a microplate reader in order to quantify extent of cell survival (FilterMax F5, Molecular Devices, USA). 2.8.3. Nitric oxide (NO) and cytokine production RAW 264.7 cells (1 × 105 cells/well) were loaded into a 12-well plate and then cultured with different concentrations of samples or LPS (1 μg/ml) for another 24 h. NO production in the conditioned media was determined based on the amount of nitrite present by the Griess reaction (Granger, Taintor, Boockvar, & Hibbs, 1996). The supernatants were collected and the production of NO, TNF-α, IL-1β and IL-6 was measured by NO assay kits and ELISA kits according to the manufacturer's protocols.

3.2. FT-IR analysis The FT-IR spectra acquired from the various polysaccharides are shown superimposed in Fig. 1. Typical polysaccharide signals can be identified at around 3385, 2915, 1645 and 1080 cm−1, all of which were found across the four samples analyzed (Shen, Zhang, & Jiang, 2017). However, there were strong absorptions at around 1784 cm−1 of DAG1 and DAG2, peaks that are not typical polysaccharide absorption peaks. The absorption wavelength of this peak is larger than the peak at 1740 cm−1 of carbonyl group on the normal acidic sugar. This may be

2.8.4. Reverse transcription and real-time quantitative PCR RAW264.7 cells were first seeded into a 6-well plate, each of which contains 1 × 106 cells diluted to 2 mL fresh culture medium. After incubation for 24 h, cells were stimulated with or without samples for an additional 24 h. Following stimulation, the cells were washed by cold phosphate-buffered saline (PBS) and the RNA was extracted using MiniBEST Universal RNA Extraction Kit according to the manufactures' procedures. The isolated RNA was used for cDNA synthesis with reverse transcrip- tase. Relative target gene quantification was conducted on a StepOnePlus Real-Time PCR system using SYBR Premix Ex Taq II kit. Reverse transcriptase amplification was conducted with an initial denaturation at 95 °C for 30 s and then 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Data was normalized to β-actin, and the primer sequences used were listed as follows: β-actin (Forward: 5′-CCCATCTACGAGGGC TAT-3′; Reverse: 5′-TGTCACGCACGATTTCC-3′), iNOS (Forward:

Table 1 Yield and monosaccharide composition of DAG1 and DAG2. Samples

a

AGW DAG1 DAG2 a b

3

Yieldb (%)

– 42.31 25.46

Relative amount (mol %) Ara

Gal

9.15 5.05 2.16

90.79 94.95 97.84

Data previously published (Tang et al., 2018). Data presented are based on the weight of AGW.

Gal:Ara

9.92 18.80 45.30

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with DAG2 bearing fewer branch points as a result of dual Smith degradation treatments. 3.4. NMR analysis To further probe for structural species of both DAG1 and DAG2 (beyond extent of degradation), 13C NMR was applied with the resultant spectra shown in Fig. 2. All the 13C NMR signals of sugar moieties were assigned completely, as showed in Table 3. Six typical signals at δ106.66, 72.85, 84.57, 71.06, 77.32 and 63.49 were found, which suggests that these belonging to C-1, C-2, C-3, C-4, C-5 and C-6 of →3)Galp-(1→ residues, respectively (Chen et al., 2017; Liu, Wen, Kan, & Jin, 2015). Compared with the carbon spectrum of the original AGW (Tang et al., 2018), there is a distinct peak at δ84.57 in the spectra of DAG1 and DAG2. This observation indicates that new sugar residues are produced during the Smith degradation. This is due to the removal of branch structures which exposed new portions of AGW's main polysaccharide chain. It was also observed that the signals at δ106.05 and 106.44 were very weak in DAG1, which were assigned to the anomeric carbons of →6)-Galp-(1→ and →3,6)-Galp-(1→, respectively. These weak anomeric carbon signals disappeared completely in DAG2, which indicates that the second Smith degradation virtually removed all the side chain residues, or at least to the point where our NMR configuration could not detect them. The attribution of these signals was consistent with the results obtained from methylation analysis.

Fig. 1. FT-IR spectra of AGW, AGS, DAG1 and DAG2.

due to the coupling effect caused by the oxidation of multiple hydroxyl groups on a single sugar molecule during smith degradation. Eventually, the absorption of the carbonyl group is blue-shifted. The absorption peak of DAG2 is significantly smaller than that of DAG1 at 1784 cm−1. This indicates that more non-sugar chains are shed over the course of two separate performance of Smith degradation. Taken together, the results of the FT-IR spectra assay proved that both DAG1 and DAG2 contained the typical groups of sugars as well as clear signs of successful Smith degradation.

3.5. Congo-red assay Polysaccharides with a triple helix conformation can form complexes with Congo red and increase the maximum absorption wavelength in aqueous or weakly alkaline solutions, and convert to single chains in highly alkaline solutions with reduced absorption wavelength (Rout, Mondal, Chakraborty, & Islam, 2008). As shown in Fig. 3, the maximum absorption wavelength of Congo red AGW and Congo red AGS decreased gradually with an increase in NaOH concentration, closing to the maximum absorption wavelength of Congo red. However, obvious shifts of maximum absorption wavelength from 498 to 512 and 514 nm were induced by the presence of the polysaccharides DAG1 and DAG2 in Congo red solution, indicating that polysaccharide-Congo red complexes had formed. At the same NaOH concentration, all the complexes showed red shifts compared with Congo red. However, it did not show the specific shift of the maximum absorption wavelength at different concentrations of NaOH. The change in the maximum absorption wavelength of DAG1 and DAG2 may be due to their adoption of random coil confirmations due to the increasing extent of Smith degradation. Similar results reported that the depolymerization of polysaccharides resulted in transformation from rigid conformations to more flexible conformations (Wang et al., 2017; Yi et al., 2012). Thus, it could be concluded that polysaccharides (DAG1 and DAG2) after Smith degradation showed rigid conformation, but not a triple-helical conformation in solution. Combined with structural information, we can speculate that the side chains of polysaccharides have a significant effect on their spatial conformation due to the clear differences induced by side chain removal.

3.3. Methylation/GC–MS analysis In order to better understand which specific glycosidic linkages were altered by Smith degradation, methylation analysis of DAG1 and DAG2 was performed. The linkage patterns and their molar ratio with respect to galactose and arabinose in both DAG1 and DAG2 are listed in Table 2. The majority of terminal units in DAG1 and DAG2 were found to be T-Galp with relative amounts of 21.8% and 10.9%, respectively. Interestingly, only a small amount of T-Araf was found in DAG1. The sugar residue with the greatest proportion was →3)-Galp-(1→, accounting for 56.2% and 79.5%, respectively. These results were completely different from the structure of the original AGW as described in our previous report (Tang et al., 2018). It was also noted that the proportion of →3,6)-Galp-(1→ dropped from 15.2% in DAG1 to 8.7% in DAG2. These results indicate that Smith degradation was capable of removing the β-1,6-galactan side chains while preserving the β-1,3galactan backbone. Although most of the short stubs of β-1,6-galactan side chains were removed, a small amount of →6)-Galp-(1→ residue did remain. In all, these results demonstrate that the key difference between DAG1 and DAG2 is the extent of removal of branching structures, Table 2 Methylation analysis of the derivatives of DAG1 and DAG2. Derivatives

2,3,4,6-Me4-Gal 2,3,4-Me3-Gal 2,4-Me2-Gal 2,4,6-Me3-Gal 2,6-Me2-Gal 2,3,5-Me3-Ara 2,3,4-Me3-Ara 2,5-Me2-Ara a b

Linkage

Galp-(1→ →6)-Galp-(1→ →3,6)-Galp-(1→ →3)-Galp-(1→ →3,4)-Galp-(1→ Araf-(1→ Arap-(1→ →3)- Araf -(1→

Molar ratioa (%) AGWb

DAG1

DAG2

3.6. Immunomodulatory activities

30.1 23.7 26.6 2.8 1.9 6.9 3.7 4.3

21.8 2.18 15.2 56.2 ND 4.5 ND ND

10.9 0.8 8.7 79.5 ND ND ND ND

3.6.1. Cell toxicity of polysaccharides on macrophages Macrophages are a type of white blood cell of the immune system that are found in all tissues to engulf and digest cellular debris, as well as anything else that does not have the type of proteins specific to healthy body cells (Ovchinnikov, 2008). Prior to investigation of the immunomodulatory activity of AGs and DAGs, their toxic effects on RAW264.7 cells were first evaluated using CCK-8 assay. As shown in Fig. 4A and B, AGs and DAGs were all nontoxic to RAW 264.7 cells and can slightly promote cell proliferation over the entire tested

Relative molar ratio, calculated from the ratio of peak areas. Data previously published (Tang et al., 2018). 4

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Fig. 2.

13

C NMR spectra of DAG1 and DAG2.

inflammation and stimulating the immune system, pro-inflammatory mediator and cytokines secreted by activated macrophages can also reduce immune responses and directly participate in defense against pathogen invasion (Mills, 2012). Therefore, the effects of AGs and DAGs on the production of NO, TNF-α, IL-1β and IL-6 in RAW 264.7 cells were investigated. As shown in Fig. 4, ELISA results showed that AGs and DAGs influenced the secretion of NO and cytokines to varying degrees. The effects of AGW and AGS on NO secretion in macrophages have been demonstrated in our previous reports (Tang et al., 2018). In comparison, AGW promoted the secretion of TNF-α and IL-6 more strongly than AGS, but weaker in IL-1β secretion. One possible explanation would be that difference in conformation between aqueous solutions of AGW and AGS caused by glucuronic acid (Tang et al., 2018), which might affect their bioactivities leading to differences in cytokine secretion. However, when the branches of the AGW were removed via Smith degradation, the activities of the polysaccharides indeed changes. Specifically, DAG1 and DAG2 were both found to enhance the secretion of NO, however in a rigorous dose-dependent manner (Fig. 4F). Compared to control group, DAG1 showed greater immune enhancement potential than that of DAG2. Moreover, DAG1 increased the secretion of TNF-α, IL-1β and IL-6 to a very high level, which might be that RAW264.7 cells initiated immune response to DAG1 rapidly and generated more cytokines (Fig. 4G-I). For example, DAG1 showed a significant activation effect on the expression of TNF-α (7914.4 ± 555.7 pg/mL) at concentrations of 800 μg/mL, which was much visible higher than that of DAG2 (2344.2 ± 642.0 pg/mL). It was also interestingly to observe that DAG1 strongly stimulated RAW264.7 secretion of IL-1β, whereas the ability of macrophages to secrete IL-β was significantly reduced in the presence of DAG2 to a level even lower than the control group (Fig. 4H). This suggests that the side chain of AGW not only affects its conformation but also plays a key role in macrophage immune stimulation. In agreement with this suggestion, it is generally believed that the triple helix structure of polysaccharides plays a key role in immune stimulation (Xu, Yan, & Zhang, 2012). Possibly, the shift to the rigid conformation also affects biological function of the DAG preparations. Previously published reports demonstrated that the side chain of polysaccharides govern their orderdisorder transition in aqueous solution (Okobira, Miyoshi, Uezu, Sakurai, & Shinkai, 2008; Yoshiba et al., 2017). This suggests that

Table 3 13 C NMR chemical shifts (ppm) for DAG1 and DAG2. Residues

C-1

C-2

C-3

C-4

C-5

C-6

T-β-D-Galp →6)-β-D-Galp-(1→ →3,6)-β-D-Galp-(1→ →3)-β-L-Galp-(1→ T-α-L-Araf

106.05 106.05 106.44 106.66 111.94

73.45 73.45 72.97 72.85 83.43

75.40 74.52 84.06 84.57 79.31

71.69 71.33 71.33 71.06 86.11

77.83 76.30 76.30 77.32 63.75

63.75 71.92 72.72 63.49

Fig. 3. Maximum absorption wavelength of polysaccharide-Congo red complex at different concentrations of NaOH.

concentration range from 25 to 800 μg/mL. Similarly, polysaccharides from other sources also have a certain role in promoting the proliferation of macrophages (Li et al., 2018; Ren et al., 2017). 3.6.2. Polysaccharides activates NO, TNF-α, IL-1β and IL-6 secretion of macrophages Macrophage activation is accepted as one of the most important events over the duration of an immune response. Beyond increasing 5

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Fig. 4. Effects of AGW, AGS, DAG1 and DAG2 on cell viability (A–B), and the production amount of NO (C) and cytokines (D–I) in RAW264.7 cells. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control group.

removal of the sidechains influenced the conformation of DAG with respect to the original AG. All of the results above suggested that partial side chain retention of AGs is beneficial for their biological activity, alluding to the structure-function relationship of AGs and the contribution which their side-chain structures provide.

focus on the effect of molecular weight on AGs activity while controlling the same side chain structure in our subsequent study.

3.6.3. Polysaccharides activates mRNA expression of iNOS, TNF-α, IL-1β and IL-6 in macrophages The effects of AGs and DAGs on mRNA expression of iNOS, TNF-α, IL-1β and IL-6 were determined using RT-QPCR. These tests were performed to further confirm and explain the results observed from ELISA testing. As shown in Fig. 5, accumulation of iNOS, TNF-α, as well as IL6, significantly increased in a dose-dependent manner when RAW264.7 cells were incubated with AGW and AGS at 100, 200, 400 and 800 μg/ mL. However, there was no obvious regularity in IL-1β mRNA expression level after treatment by AGW. Similarly, the mRNA expression of iNOS, TNF-α, IL-1β and IL-6 were also promoted by DAG1 in a dosedependent manner. However, DAG2 only showed a promoting effect toward IL-6 mRNA expression. From this it appears to be concentrationdependent inhibition for iNOS, TNF-α and IL-1β. This suggests that DAG2 does not contribute to the stimulation of macrophage cytokines, consistent with the results of the ELISA. In conclusion, AGW and AGS may up-regulate the production of NO, TNF-α and IL-6 through promoting the gene expression of iNOS, TNF-α and IL-6. Furthermore, this effect is still present when removing some of the side chains (DAG1), but this stimulating effect drops dramatically when the side chain structures are extensively severed (DAG2). The data suggests that the immunological activity of polysaccharides, AG in particular, depends not only on the type of monosaccharide, molecular weight and spatial conformation, but also on the degree of branching. Next, we intend to

In this study, the immunomodulatory activity of two arabinogalactans (AGW and AGS) from larch was demonstrated, while the change in immunological activity was compared by removing part of the AG side chain. In vitro bioactivity tests showed that AGW and AGS possessed notable immunomodulatory activities that may be mediated through the promotion of the enhancement of NO, TNF-α, IL-1β and IL6 production. Partial removal of the side chain promoted immunomodulatory activity, but excessive removal results in a sharp decrease in immunomodulatory activity. These results provide new momentum for the development of future polysaccharide immunoassays. Future work should seek to analyze the structure-function relationship of polysaccharides in even greater detail. This suggestion is not limited to arabinogalactans, but rather all branched polysaccharides that could be extracted from other resources.

4. Conclusions

Acknowledgments This work was supported by the National Key R&D Program of China (2016YFD0600803) and State Key Laboratory of Pulp and Paper Engineering (201812). The authors thank the Project of First-Class Discipline and the Doctorate Fellowship of Nanjing Forestry University for supporting the work presented in this paper.

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Carbohydrate Polymers 227 (2020) 115354

S. Tang, et al.

Fig. 5. Stimulations of AGW, AGS, DAG1 and DAG2 on mRNA expression in RAW264.7 macrophages. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control group.

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