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Chemical and Biological Technologies in Agriculture
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The polysaccharide from Aralia continentalis Kitagawa enhances immune responses via activating the MAPKs and NF-κB signaling pathways in RAW 264.7 macrophages

Chemical and Biological Technologies in Agriculture volume 11, Article number: 145 (2024) Cite this article

Abstract

Background

Polysaccharides derived from Aralia continentalis Kitagawa possess excellent biological properties, such as anti-tumor, antioxidant, antibacterial, lipid-lowering, and anti-inflammatory. However, the immunomodulatory effects of these polysaccharides on macrophages and their underlying mechanisms remain largely unexplored due to their complex molecular structure.

Results

The study isolated and characterized a pure polysaccharide, namely WACP(S)-A3-b from Aralia continentalis Kitagawa to investigate its impact on RAW 264.7 cell activation. The structural analysis of WACP(S)-A3-b revealed an average molecular weight of 40.1 kDa with a pectin-like structure composed of HG and RG-I domains, primarily composed of galacturonic acid, rhamnose, galactose, fucose, and arabinose at molar ratios of 55.56: 19.60: 10.29: 7.85: 6.69; NMR found that WACP(S)-A3-b contains α-1,4-GalpA, α-1,2-Rhap, α-1,2,4-Rhap, and t-α-GalpA. Further results demonstrated that the immunomodulatory activity of WACP(S)-A3-b could enhance the production of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), and promote the expression of interleukin-1beta (IL-1β). Additionally, WACP(S)-A3-b could activate MAPKs and NF-κB signaling pathways, thereby enhancing the ability of RAW 264.7 macrophages to release cytokines.

Conclusions

The study isolated and purified the Aralia continentalis Kitagawa stem polysaccharide, clarified the basic structure of the polysaccharide, and explored the mechanism of immune activity, which provided a theoretical basis for the structure–activity relationship of the polysaccharide.

Graphical abstract

Introduction

Aralia continentalis Kitagawa, belonging to the Araliaceae family, is an herbaceous plant widely distributed in East Asia, including northern China, the Korean Peninsula, and Japan [1,2,3]. Aralia continentalis Kitagawa has been known for its potential therapeutic effects, including dispelling wind and dampness, promoting blood circulation, diuresis, detoxification, and relieving fever and pain [4]. Previous studies have discovered numerous active substances in Aralia continentalis Kitagawa, such as flavonoids, saponins, polysaccharides, alkaloids [5,6,7]. Among them, polysaccharides have been reported to exhibit various biological activities, such as anti-tumor [8], immune regulation [9], antioxidant [10], and improvement of the intestinal microenvironment [11]. However, the uncertain structure and lack of clear structure–activity relationships of these polysaccharides significantly limits further study on their mechanisms of action and developing novel products or drugs.

Mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) are two major signaling pathways associated with macrophage activation and regulating the secretion and maturation of immune molecules and related cytokines. MAPKs are serine/threonine protein kinases found in most eukaryotes, including three family members c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases (ERK), and protein kinase-38 (p38), and can be activated by the transforming growth factor-beta (TGF-β)-activated kinase 1 (TAK1) [12]. It is reported that subsequent activation of MAPKs could promote the translocation of activating protein 1 (AP1) from the cytoplasm to the nucleus, thereby regulating mRNA transcription by binding to the promoter region of relevant genes and leading to the secretion and maturation of cytokines and immune molecules, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β, and IL-6 [13,14,15,16]. NF-κB belonging to the family of transcription factors is mainly associated in the regulation of inflammatory responses, immune responses, and cell proliferation [17]. NF-κB forms a complex with its inhibitor protein IκB, preventing its entry into the nucleus. IκB is degraded upon stimulation by inflammatory factors, bacterial infection, or cellular stress, leading to the release and nuclear translocation of NF-κB [18]. The activated NF-κB can bind to specific DNA sequences in the nucleus, promoting the transcription of target genes. These target genes encode various inflammatory factors, cytokines, anti-apoptotic proteins, and other molecules involved in regulating various cellular physiological functions [19]. Therefore, NF-κB plays an inevitable role in inflammation, immune regulation, cell proliferation, and cell survival. Polysaccharides could induce the secretion of NO, TNF-α, IL-6 and IL-1β by activating the MAPKs/NF-kB signaling pathways, and then activate RAW264.7 macrophages [20].

In this study, a more in-depth investigation of the polysaccharides derived from Aralia continentalis Kitagawa was conducted in terms of systematic separation, purification, structural characterization, and their immunomodulatory mechanisms. The experimental results provide a scientific basis for the structural analysis and development of polysaccharides from Aralia continentalis Kitagawa.

Materials and methods

Materials

Aralia continentalis Kitagawa was supplied by the College of Landscape Architecture, Changchun University, Changchun, China. DEAE-cellulose bought from the Shanghai Chemical Reagent Research Institute, and Sephadex G-100 was acquired from Pharmacia Biotech. The RAW 264.7 murine macrophage cell line was obtained from the American Type Culture Collection. High-glucose DMEM complete medium and fetal bovine serum were purchased from HyClone (USA). Lipopolysaccharide (LPS) was obtained from Sigma (USA). ELISA Kits bought from Elabscience Biotechnology Co., Ltd. All antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd. Standards of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, fucose and the other reagents were commercially available or produced in China.

Extraction and purification of the polysaccharide

The stem of Aralia continentalis Kitagawa was first extracted with distilled water at 90 °C for 2 h (1:20 w/v), and the extraction was repeated twice under the same conditions. The extract was concentrated under vacuum at 60 ℃ and diluted to a final concentration of 70% by adding 95% ethanol to precipitate the polysaccharide. The precipitate was collected by centrifugation (4000 rpm, 20 min) and lyophilized [21]. The extracted water-soluble polysaccharide was named WACP(S). WACP(S) was dissolved with dH2O, centrifuged (4000 rpm 15 min), and separated by DEAE-Cellulose (Cl−, 1.5 × 14 cm). After elution with distilled water and different concentrations of NaCl, the neutral polysaccharide part (WACP(S)-N) and five acidic parts (WACP(S)-Ax) were obtained [22]. The homogeneous component WACP(S)-A3-b was obtained by further purification of WACP(S)-A3 by Sephadex G-100 gel permeation chromatography.

Chemical composition analysis

The total carbohydrate content of the samples was determined by the phenol–sulfuric acid method using glucose content as the standard [23]. Bovine serum albumin was used as a reference for the determination of protein content in samples by the Bradford method. The m-hydroxydiphenyl method was used to determine the uronic acid content [24].

Analysis of monosaccharide composition and molecular weight homogeneity

WACP(S)-A3-b (2 mg) was hydrolyzed by adding 1 M hydrochloric acid to anhydrous methanol at 80 ℃ for 12 h, followed by 2 M trifluoroacetic acid (TFA) at 120 ℃ for 1 h. Finally, 1-phenyl-3-methyl-5-pyrazolone (PMP) was pre-column derivated and the monosaccharide composition was determined by HPLC [25]. High-performance gel permeation chromatography (HPGPC) with a TSK gel G-3000 PWXL column (7.8 × 300 mm) (Tosoh Corporation, Japan) and Shimadzu (Kyoto, Japan) HPLC systems were used in combination to determine molecular weight [26].

Fourier transform infrared spectroscopy (FT-IR) analysis

The samples were ground uniformly with potassium bromide (KBr) powder, and the spectral changes in the frequency range of 4000–400 cm−1 were measured by FT-IR [27].

NMR analysis

WACP(S)-A3-b (20 mg) dissolved in 0.5 mL D2O, and the precipitate was removed by centrifugation. 1H, 13C, HSQC and HMBC NMR spectra of polysaccharide samples were determined using a Bruker Avance NMR spectrometer (Germany) with acetone as an internal standard. A Bruker 5 mm broadband probe was run at 600 MHz for 1H NMR and 150 MHz for 13C NMR. The detection temperature was 20 ℃ [28].

Cell culture

RAW 264.7 cells were cultured in DMEM high glucose complete medium (containing 1% penicillin and streptomycin) and placed in 5% CO2 incubator at 37 °C. The cells were passaged when the bottom 80% (volume fraction) of the culture bottle was covered [29]. To prevent cells differentiation, cells were blown directly at the bottom of the dish with a 1 mL pipetting gun to replace enzyme digestion during cells passage.

Cell viability assay

RAW 264.7 cells were seeded in 96-well plates at a density of 1.5 × 105 cells/well, cultured overnight, and treated with different concentrations of WACP(S)-A3-b (50, 100, 200, 400, 600, 800 μg/mL) or LPS (1 μg/mL) for 24 h. The cell viability was detected by CCK-8 assay [30].

Measurement of NO

The 1.5 × 105 cells/well cell suspension was seeded in a 96-well plates (100 µL/ well) and placed in an incubator overnight. After cells adherence, 200 µL DMEM high glucose complete medium with different mass concentrations of WACP(S)-A3-b (0, 50, 100, 200, 400, 800 μg/mL) was added to each well of the sample group, and 200 µL DMEM high glucose complete medium containing LPS (1 μg/mL) was added to each well of the positive control group. Each group was incubated for 24 h in triplicate Wells. Three replicates were performed. 50 µL of 1% sulfonamide and 0.1% naphthylamine were added to 100 µL of cells supernatant in turn. After 20 min at room temperature, the absorbance value at 540 nm was detected by a multifunctional microplate reader.

Enzyme-linked immunosorbent assay

RAW 264.7 cells were seeded in 96-well plates at a density of 1.5 × 105 cells/well which were counted by the microscopic counting method and cultured overnight at 37 ℃. The sample was added to the plate and incubated for 24 h. The supernatant was collected by centrifugation, and the related immunomodulatory molecules were detected by enzyme-linked immunosorbent assay. The concentrations of TNF-α, IL-1β, and IL-6 in the cell supernatant were detected according to the instructions of ELISA kit.

Western blotting

RAW 264.7 cells (1 × 106 cells/well) were treated as described in “Enzyme-linked immunosorbent assay” section. The cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer on ice for 30 min, and the proteins were extracted and quantified by the BCA Kit. Detergent-insoluble material was removed, and equal amounts of protein were fractionated by 10% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and then transferred onto the polyvinylidene fluoride (PVDF) membranes. At the end of the membrane transfer, immerse PVDF membrane in blocking solution in a horizontal shaker at room temperature for 1 h. At the end of closure, the blocking solution was discarded, the PVDF membrane was cut as required and then incubated with appropriately diluted 5% primary antibody for 2 h in 5% BSA. The membranes were then incubated for an additional 1 h with goat antirabbit IgG/horseradish peroxidase (HRP). signals were detected by enhanced chemiluminescence.

Inhibition experiments

RAW 264.7 cells were pretreated with p38 inhibitor SB203580 (5 µmol/L) or NF-κB inhibitor BAY 11–7082 (2.5 µmol/L) for 2 h and then co-treated with WACP(S)-A3-b for 30 min. The expression of related proteins was detected as described in “Western blotting” section [31].

Statistical analysis

All tests were performed in three parallel sets of experiments and the divergences were presented as mean ± SD, differences between the groups were analyzed through t tests analysis, P < 0.05, P < 0.01, and P < 0.001, respectively, represented significant difference, extremely significant difference and highly significant difference.

Results

Extraction and purification of polysaccharide from Aralia continentalis Kitagawa

The water-soluble polysaccharide (WACP(S)) was extracted from the stem of Aralia continentalis Kitagawa (Fig. 1A), with a yield of 12.90 ± 1.13% in relation to the dry weight of the initial material. WACP(S) was fractionated into a neutral polysaccharide fraction WACP(S)-N (yield 19.45 ± 1.22%) by ion-exchange chromatography, and five acid polysaccharide fractions, namely WACP(S)-A1 (yield 6.18 ± 0.23%), WACP(S)-A2 (yield 25.83 ± 0.21%), WACP(S)-A3 (yield 33.90 ± 2.35%), WACP(S)-A4 (yield 5.05 ± 0.17%), and WACP(S)-A5 (yield 3.58 ± 0.12%) were obtained (Fig. 1B, Table 1). WACP(S)-A3 was further purified with gel permeation chromatography to obtain a major homogeneous fraction WACP(S)-A3-b (yield 41.82 ± 0.22%) (Fig. 1C). The standard curve of molecular weight distribution was drawn using dextran standards of different molecular weights (1 kDa, 5 kDa, 12 kDa, 25 kDa, 50 kDa) and the linear regression equations y = -0.4524x + 9.5477, R2 = 0.9945. The molecular weight of WACP(S)-A3-b was around 40.1 kDa, which was determined by HPGPC (Fig. 1D). The monosaccharide composition analysis showed that WACP(S)-A3-b contained galacturonic acid as the main component, followed by rhamnose, galactose, fucose, and arabinose in minor quantity (Table 2), and at molar ratios of 55.56:19.60:10.29:7.85:6.69.

Fig. 1
figure 1

Extraction and purification of polysaccharides. A Extraction of water-soluble polysaccharide (WACP) from Aralia continentalis Kitagawa. B WACP(S) was separated by DEAE-cellulose anion-exchange chromatography to produce WACP(S)-N and WACP(S)-A1, A2, A3, A4, A5. C WACP(S)-A3 was purified by Sephadex G-100 column to produce WACP(S)-A3-b. D Elution profile of WACP(S)-A3-b on HPGPC

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Table 1 The yields and chemical compositions of different WACP(S) grades
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Table 2 Monosaccharide compositions of different WACP(S)-A3 grades
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Primary structure analysis of WACP(S)-A3-b

FT-IR analysis

The key functional groups of WACP(S)-A3-b were identified by FT-IR spectroscopy. As shown in Fig. 2, two characteristic absorptions of the polysaccharide were observed at 3442.04 cm−1 and 2947.54 cm−1, corresponding to the O–H stretching of carbohydrates and C–H stretching vibrations of the methyl or aromatic groups, respectively [21]. The absorption peak which is near 1735.42 cm−1 and 1617.12 cm−1 corresponds to the carboxyl group vibration of methyl-esterified and free COO-, suggesting the presence of uronic acid in the sample [32]. The band near 1420.56 cm−1 was caused by the bending vibration of C–H [33]. The relatively weak absorption peaks at 1098.40 cm−1 and 1012.25 cm−1 refer to the stretching vibrations of C–O, suggesting the existence of a pyranose ring [34]. The results indicated that WACP(S)-A3-b has the characteristic absorption peak of acidic polysaccharide with partial esterification.

Fig. 2
figure 2

FT-IR spectrum of WACP(S)-A3-b

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NMR analysis

The main chemical shifts of NMR spectra from WACP(S)-A3-b are illustrated in Fig. 3 and Table 3. The signals of WACP(S)-A3-b were mainly distributed in the region of 3.0–5.5 ppm (1H-NMR, Fig. 3A) and 60–110 ppm (13C-NMR, Fig. 3B), representing the typical distribution of NMR signals from polysaccharides [35]. The signals at 98.82, 68.23, 69.85, 77.14, 71.27, and 174.99 ppm correspond to the C-1, C-2, C-3, C-4, C-5,and C-6 of α-1,4-GalpA.The signals at 5.02, 3.84, 3.98, 4.35, and 4.58 ppm correspond to the H-1, H-2, H-3, H-4, and H-5 of α-1,4-GalpA [36]. The signals at 20.07 ppm in the acetyl groups were attached to the α-D-GalpA units, confirming the esterification of HG-type pectin in WACP(S)-A3-b. In addition to the characteristic signal peaks of HG-type pectin, some characteristic signal peaks of RG-I pectin were found, such as the chemical shift representing the C-6 characteristic signal peak of α-1,2-Rhap at 16.37 ppm [21, 37]. Based on the combined results of 2D-NMR, including HSQC spectra (Fig. 3C) and HMBC spectra (Fig. 3D), and data from references, the anomeric proton and carbon signals at δ 5.02/98.82 ppm, 5.20/98.21 ppm, 4.94/97.40 ppm, and 5.02/98.89 ppm were assigned to α-1,4-GalpA, α-1,2-Rhap-, α-1,2,4-Rhap, and t-α-GalpA, respectively. Based on the monosaccharide composition of WACP(S)-A3-b, it can be inferred that WACP(S)-A3-b is a pectin-like polysaccharide composed of HG and RG-I domains.

Fig. 3
figure 3

NMR spectrum of WACP(S)-A3-b. A 1H spectrum; B 13C spectrum; C HSQC spectrum; D HMBC spectrum

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Table 3 Chemical shifts of the 13C and 1H NMR spectrum of WACP(S)-A3-b
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Effect of WACP(S)-A3-b on RAW 264.7 cell viability

RAW 264.7 cells were treated with WACP(S)-A3-b at different concentrations of 0, 50, 100, 200, 400, and 800 µg/mL to investigate the cytotoxic effects. As shown in Fig. 4A, different mass concentrations of WACP(S)-A3-b had varying effects on the cell viability of RAW 264.7 cells compared to the blank control group. When the mass concentration was lower than 600 μg/mL, with the increase of mass concentration, the cell viability gradually increased and then decreased. However, at a mass concentration of 800 μg/mL, the cell viability showed a highly significant decrease (P < 0.05), indicating that high mass concentrations of the polysaccharide sample had a certain level of toxicity, reducing cell viability. Based on the overall cell viability experimental results, the mass concentrations of 50, 100, and 200 μg/mL were selected for subsequent experimental treatments, and the immunomodulatory effects of WACP(S)-A3-b on RAW 264.7 cells were evaluated.

Fig. 4
figure 4

Effect of WACP(S)-A3-b on cell viability, NO production, and cytokine expression and secretion in RAW 264.7 cells. A Cell viability of RAW 264.7 cells treated with WACP(S)-A3-b at various concentrations; B NO production; C, D, and E secretion of cytokines. Versus the negative control group. Data are presented as means ± SD. *P < 0.05; ***P < 0.001

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WACP(S)-A3-b increased NO production in RAW 264.7 cells

Massive release of NO is an indication of macrophage activation. In this study, the effect of WACP(S)-A3-b on the immune processes of macrophages was determined by a Griess assay. The NO concentration of positive control group (LPS) was 88.65 ± 1.76 μM, which was significantly higher than that of control group, suggesting the data were valid (P < 0.001). WACP(S)-A3-b could improve the release of NO, with the largest NO concentration at 200 μg/mL, indicating that WACP(S)-A3-b may have a pro-inflammatory effect (Fig. 4B). Therefore, 200 μg/mL was selected for the subsequent experimental studies.

Effects of WACP(S)-A3 on the secretion of immunomodulatory factors in RAW 264.7 macrophages

The secretion of immunomodulatory factors is closely related to the degree of immunological response in macrophages. TNF-α, IL-1β, and IL-6 are three major immunomodulatory factors in macrophages stimulated by immunogens [38]. The present study has proved that 200 μg/mL of WACP(S)-A3-b could effectively enhance the secretion of NO. Therefore, the RAW 264.7 cells were treated with 200 μg/mL of WACP(S)-A3-b, and the concentrations of TNF-α, IL-1β, and IL-6 in the cell supernatant were measured to assess the level of macrophage activation. As shown in Fig. 4(C-E), the secretion levels of TNF-α, IL-1β, and IL-6 in the blank control group were 161.64 pg/mL, 137.06 pg/mL, and 782.15 pg/mL, respectively. Compared to the blank control group, the positive control group exhibited a highly significant increase in the mass concentrations of TNF-α, IL-1β, and IL-6 (P < 0.001). Similarly, compared to the blank control group, the experimental group showed a highly significant increase in the mass concentrations of TNF-α, IL-1β, and IL-6 (P < 0.001). At a polysaccharide concentration of 200 μg/mL, the secretion levels of TNF-α, IL-1β, and IL-6 were 458.59 pg/mL, 484.70 pg/mL, and 2662.15 pg/mL, respectively. These results indicated that a mass concentration of 200 μg/mL of WACP(S)-A3-b could promote the secretion of cytokine.

Effects of WACP(S)-A3-b on the activation of MAPKs and NF-κB signaling pathways

The MAPKs signaling pathway

RAW 264.7 cells were treated with 0, 50, 100, and 200 µg/mL of WACP(S)-A3-b for 30 min and subjected to Western blot analysis to investigate the relationship between WACP(S)-A3-b-induced activation of RAW 264.7 macrophages and the MAPKs pathway. As shown in Fig. 5(A-D), different mass concentrations of WACP(S)-A3-b could promote the phosphorylation of JNK, ERK, and p38. Moreover, the phosphorylation showed a dose-dependent pattern, with increasing mass concentrations of WACP(S)-A3-b leading to increase in the phosphorylation levels (relative expression of p-JNK/JNK, p-ERK/ERK, and p-p38/p-38). When the mass concentration was 200 µg/mL, the phosphorylation level reached the highest, with JNK, ERK and p38 phosphorylation levels nearly being 2.5, 1.5, and 2.5 times higher than those in the blank control group. This result indicates that WACP(S)-A3-b could activate macrophages by promoting the phosphorylation of JNK, ERK, and p38. Furthermore, the RAW 264.7 cells were pretreated with p38 inhibitor SB203580, and then co-treated with WACP(S)-A3-b to determine the expression levels of proteins. According to results of Western blot and relative gray value analysis of protein expression (Fig. 5E, F), the addition of p38 inhibitors significantly reduced protein expression (P < 0.01)). Overall, these results suggest that WACP(S)-A3-b could activate macrophages via activating the MAPKs signaling pathway.

Fig. 5
figure 5

Effects of WACP(S)-A3-b on MAPKs signaling pathways. A–C Enzyme-linked immunosorbent assay analysis of p-JNK/JNK, p-ERK/ERK and p-p38/p38. D Western blot analysis of p-JNK/JNK, p-ERK/ERK and p-p38/p38 in RAW 264.7 cells. E, F Expression of p-p38/p38 after co-treatment of RAW 264.7 cells with WACP(S)-A3-b and SB203580 for 30 min. Data are presented as mean SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001

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The NF-κB signaling pathway

Herein, the relationship between WACP(S)-A3-b-induced activation of RAW 264.7 macrophages and the NF-κB signaling pathway was investigated. The RAW 264.7 cells were treated with 0, 50, 100, and 200 µg/mL of WACP(S)-A3-b for 30 min, and then subjected to Western blot analysis. As shown in Fig. 6A–C, an increasing mass concentration of WACP(S)-A3-b was accompanied by a gradual increase in the extent of IκB-α degradation. Following treatment with 200 μg/mL WACP(S)-A3-b for 30 min, only 31.46% of IκB-α remained undegraded, with the majority of IκB-α undergoing degradation. Later, the macrophages were pretreated with BAY 11–7082 and subsequently co-treated with WACP(S)-A3-b to investigate the role of the NF-κB signaling pathway in activating RAW 264.7 macrophages induced by WACP(S)-A3-b. The analytical results of relative grayscale intensities of protein expression levels from Western blot (Fig. 6D–F) revealed reduction in the extent of IκB-α degradation. Collectively, these findings indicate that WACP(S)-A3-b could promote macrophage activation through the NF-κB signaling pathway [39].

Fig. 6
figure 6

Effects of WACP(S)-A3-b on NF-κB signaling pathways. A, B Enzyme-linked immunosorbent assay analysis of NF-κB and IκB-α. C Western blot analysis of NF-κB and IκB-α in RAW 264.7 cells. D–F Expression of NF-κB and IκB-α after co-treatment of RAW 264.7 cells with WACP(S)-A3-b and BAY 11–7082 for 30 min. Data are presented as mean SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001

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Discussion

Various substances such as polysaccharides, proteins, polyphenols and flavonoids in natural plants have been shown to be bioactive [40]. As the main natural component in plant, the biological activities of polysaccharides are related to their structure, including molecular weight, monosaccharide composition, glycosidic linkage, α/β configuration, conformation, and branching degree [41, 42]. Polysaccharides can affect immunomodulatory activity in many ways [43, 44]. Particularly, the immunomodulatory activity is influenced by monosaccharide composition and molecular weight [45]. In this study, WACP(S)-A3-b had a molecular weight of 40.1 kDa and was composed of five monosaccharides, with the majority being galacturonic acid (55.56%) and rhamnose (19.60%), resulting in its high viscosity and uronic acid/carbohydrate ratio, FT-IR and NMR results show that WACP(S)-A3-b contains HG and RG-I domains. In another study, galacturonic acid, rhamnose, and galactose were the main monosaccharides of polysaccharides from the root tubers of Aralia continentalis Kitagawa, with some differences in proportions [21]. Furthermore, previous reports have reported that uronic acid in polysaccharides plays a crucial role in affecting their anti-tumor activity [46, 47]. The present study results revealed the presence of a high content of galacturonic acid in WACP(S)-A3-b, partially explaining its anti-tumor and immunomodulatory functions.

The immunomodulatory activity was considered one of the most significant biological properties of polysaccharides, as this function could directly or indirectly influence other biological activities, such as antioxidant, anticancer, and antiviral effects [48].

Therefore, the role of immunomodulatory effects of WACP(S)-A3-b in activating the capacity of murine macrophages was investigated. The results showed that WACP(S)-A3-b could enhance the capacity of releasing cytokines from RAW 264.7 cells, an effect that has not been reported in other literature for polysaccharides isolated from Aralia continentalis Kitagawa. Hye et al. demonstrated that the ethyl acetate fraction of Aralia continentalis Kitagawa root increased NO production in a dose-dependent manner and inhibited lipopolysaccharide levels, showing therapeutic potential in inflammation [22]. Similarly, Xie et al. demonstrated that a novel polysaccharide isolated from alfalfa enhanced the pinocytic and phagocytic capacity of RAW 264.7 cells and increased the secretion of NO, TNF-α, IL-1β, and IL-6 [49]. These findings were consistent with the present study results.

Furthermore, the protein expression in the MAPKs and NF-κB signaling pathways was investigated by treating the RAW 264.7 macrophage cells with different doses of WACP(S)-A3-b. The Western blot results showed that WACP(S)-A3-b upregulated the total protein levels of JNK, ERK and p38 and the phosphorylation levels of p-ERK, p-JNK, and p-p38, indicating that WACP(S)-A3-b could promote the production of immunomodulatory factors by activating the MAPKs signaling pathway. This finding was consistent with the previous study results, reporting that polysaccharides from alfalfa and Tinospora cordifolia could activate macrophages by upregulating the p-JNK, p-ERK, and p-p38 levels. Additionally, the role of WACP(S)-A3-b in activating the NF-κB signaling pathway was investigated. The Western blot results showed that WACP(S)-A3-b could effectively promote the degradation of IκB-α. This finding was consistent with the previous studies, reporting that yam polysaccharides could activate macrophages through the NF-κB pathway, and polysaccharides from Acanthopanax senticosus and Ecklonia cava could activate the B cells through the NF-κB signaling pathway [50]. These results confirm that WACP(S)-A3-b can activate the RAW 264.7 cells through the MAPKs and NF-κB signaling pathways. Consequently, these discoveries suggest that WACP(S)-A3-b may serve as a promising immunostimulant, thus holding potential applications in the fields of functional foods and pharmaceuticals [51].

Conclusion

In summary, WACP(S)-A3-b obtained through hot water extraction and ethanol precipitation was mainly composed of galacturonic acid, rhamnose, galactose, fucose, and arabinose, with a pectin-like structure composed of HG and RG-I domains. The results showed that WACP(S)-A3-b activated RAW 264.7 cells by enhancing cell viability and promoting the secretion of NO, TNF-α, IL-1β, and IL-6. Furthermore, WACP(S)-A3-b promoted the phosphorylation of MAPKs-related proteins, including JNK, ERK, and p38, and the degradation of IκB-α in the NF-κB pathway. These data contribute to a better understanding of the molecular mechanisms underlying the activation of macrophages by WACP(S)-A3-b. This study lays a foundation for exploring and utilizing WACP(S)-A3-b as a novel immunomodulator in functional foods and the pharmaceutical industry. Nevertheless, further research is warranted to investigate the in vivo regulatory effects of WACP(S)-A3-b on macrophages, particularly within the context of the tumor immune microenvironment and their underlying mechanisms.

Availability of data and materials

No datasets were generated or analyzed during the current study.

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This research was funded by the Science and Technology Projects of Jilin Provincial Department of Education (JJKH20210617KJ) and the Science and Technology Development Project Foundation of Jilin Province (20210203179SF).

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Authors and Affiliations

  1. College of Food Sciences and Engineering, Jilin Agricultural University, Changchun, 130018, China

    Xi Wang, Di Xie, Hewen Hu & Dawei Wang

  2. College of Food Sciences and Engineering, Changchun University, Changchun, 130024, China

    Xi Wang, Liyang Liu & Tianyu Wang

  3. Jilin Province Product Quality Supervision and Inspection Institute, Changchun, 130012, China

    Xu Zhang & Siqi Wang

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Contributions

W.X.: conceptualization, data curation, investigation, methodology, writing—original draft; L.L.: formal analysis, methodology, visualization, writing—original draft; X.Z.: data curation, methodology, software; D.X.: methodology, formal analysis; H.H.: conceptualization, data curation, investigation; S.W.: methodology, writing—review and editing; D.W.: conceptualization, resources, writing—review; T.W.: funding acquisition, supervision. All authors have read and agreed to the published version of the manuscript.

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Wang, X., Liu, L., Zhang, X. et al. The polysaccharide from Aralia continentalis Kitagawa enhances immune responses via activating the MAPKs and NF-κB signaling pathways in RAW 264.7 macrophages. Chem. Biol. Technol. Agric. 11, 145 (2024). https://doi.org/10.1186/s40538-024-00649-y

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