Immunomodulation of RAW264.7 cells by CP80-1, a polysaccharide of Cordyceps cicadae, via Dectin-1/Syk/NF-κB signaling pathway

ABSTRACT

Cordyceps cicadae is a rare edible and medicinal entomogenous fungus. Here, the structural characteristics and immunomodulatory activity of C. cicadae polysaccharide CP80-1 were preliminarily elucidated. It showed that CP80-1 of 250.431 kDa was mainly composed of glucose, xylose and rhamnose. Physiologically, CP80-1 can regulate the immune of RAW264.7 cells by promoting the proliferation, phagocytosis, and secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)−1β, and interleukin (IL)−6. However, these effects were significantly attenuated when Dectin-1 receptor expression was inhibited by laminarin. Moreover, laminarin down-regulated the mRNA levels of relevant genes, inhibited CP80-1-driven expression of proteins related to spleen tyrosine kinase (Syk) and nuclear factor kappa-B (NF-κB) signaling pathway. Additionally, CP80-1 induced the nuclear translocation of NF-κB subunit p65 in RAW264.7 cells. These results suggest that CP80-1 may activate RAW264.7 cells by regulating the Dectin-1/Syk/NF-κB signaling pathway, which provides theoretical basis for the development of C. cicadae in immunomodulation.

KEYWORDS:

• Immunomodulatory
• polysaccharide
• Cordyceps cicadae
• macrophage
• Dectin-1

1. Introduction

Cordyceps cicadae is a rare entomogenous fungus complex formed by a fungus of Claricipitaceae parasitized on the cicada nymph, which has high nutritional value and extensive pharmacological effects (Liu, 2012). C. cicadae has been used as medicine and tonic for over 1600 years and was the first medicinal specie of Cordyceps (Nxumalo et al., 2020), mainly for the treatment of chronic kidney diseases, dispelling wind and heat, palpitations, and children with seizures (Hsu et al., 2015; Zeng et al., 2021). Modern research displayed that the main active ingredients of C. cicadae are polysaccharides, nucleosides, cordycepin, ergosterol and its peroxides, N6-(2-hydroxyethyl) adenosine, etc (Hsu et al., 2015; Li et al., 2021). Among these, polysaccharides represent one of the most abundant components in C. cicadae and are considered to be mainly responsible for the pharmacological activities, including immunomodulatory, neuroprotection, antioxidant, renal protection and anti-aging activity (Deng et al., 2020; Olatunji et al., 2016a; Olatunji et al., 2016b; Xu et al., 2018; Zhu et al., 2020). Immunomodulation is an important biological function of fungal polysaccharides, and they can trigger the non-specific response of the body's immune system to viruses, bacteria, and other foreign antigens, and even tumor cells (Giavasis, 2014; Guo et al., 2009). Our previous study found that polysaccharides from C. cicadae could promote the maturation of dendritic cells (DCs) and display excellent immunomodulatory activity (Feng, 2016), but the underlying mechanism has not been explored.

Macrophages are monocyte-derived immune cells that act as a bridge between the specific and non-specific immune responses of the body (Schepetkin & Quinn, 2006; Zhao et al., 2018). Studies have shown that there are a variety of fungal polysaccharides receptors on the surface of macrophages, such as complement type 3 receptor (CR3), scavenger receptor (SR), mannose receptor (MR), Dectin-1 receptor (Dectin-1) and Toll-like receptor (TLR) (Gao et al., 2020; Guo et al., 2021; Lee et al., 2015). When fungal polysaccharides bind to these receptors, macrophages can alter cellular activity via downstream pathways such as mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3 K)/Akt, and nuclear factor kappa B (NF-κB). Furthermore, it secretes tumor necrosis factor (TNF)-α, interleukin (IL)−1β and IL-6(Ren et al., 2021; Wang et al., 2017b). It was found that polysaccharide JCH-1 of C. cicadae regulates the immune modulatory activity of RAW 264.7 cells via TLR4/MAPK/NF-B signals (Xu, 2019). However, the mechanism of polysaccharides from C. cicadae on other receptor-mediated signaling pathways remains unclear.

In this study, a water-extracted polysaccharide named CP80-1 was isolated and purified from C. cicadae. The preliminary chemical properties of CP80-1 were characterized. In addition, the RAW264.7 macrophage model was used to investigate its immunomodulatory activity and underlying mechanisms. This study found that CP80-1 could regulate Dectin-1 receptor expression, and induce Syk/NF-κB signaling pathway, showing its activation of RAW264.7 macrophages. These results help to further elucidate the biological activities of C. cicadae polysaccharides and develop them into functional foods or drugs with immunomodulatory activities.

2. Materials and methods

2.1. Materials and reagents

C. cicadae was collected from Jurong, Jiangsu Province, P. R. China. It was identified as C. cicadae by Professor Zhen Ouyang. 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit, NO assay kit and NF-κB activation-nuclear transport assay kit were supplied by Beyotime Biotechnology (Shanghai, China). Lipopolysaccharide (LPS) was purchased from MultiSciences (Lianke) Biotechnology Co., Ltd. (Zhejiang, China). ELISA kits from Shanghai Yubo Biotech Co., Ltd. (Shanghai, China). Trizol, reverse transcription kit and quantitative PCR kit were supplied by Sangon Biotech Co., Ltd. (Shanghai, China). Laminarin was purchased from Sigma (MO, USA). Dectin-1, Syk, IKKα/β, p-IKKα/β and IκBα antibodies were obtained from CST (Beverly, USA). As for the other chemicals and reagents, they were all analytical grade.

2.2. Extraction and purification of CP80-1

CP80-1 was prepared from C. cicadae based on our previously reported method (Wang et al., 2014a). Shortly, the dried C. cicadae power (100.0 g) was defatted with petroleum ether. The defatted dried C. cicadae powder was thoroughly soaked with water at the ratio of 1:10 and twice extracted at 90°C under reflux for 2 h. The extract was centrifuged, filtered, and the supernatant was collected and added to a concentration of 80% ethanol and placed at 4°C for 12 h. Then centrifuged and freeze-drying it to obtain the crude polysaccharides. Savage reagent was used to remove proteins and activated carbon was adsorbed and decolorized from the crude polysaccharides. The crude polysaccharides were then eluted using distilled water, and 0.1, 0.2 and 0.3 mol/L NaCl solution at 1 mL/min on DEAE cellulose-52 column. A phenol-sulfuric acid method was used to determine the carbohydrate content of the elution fractions.(DuBois et al., 1956). The distilled water eluted fraction was purified by Sephadex G-100 column to obtain CP80-1.

2.3. Characterization analysis of CP80-1

2.3.1. Chemical composition analysis

The total carbohydrate content of CP80-1 was detected using an improved phenol-sulfuric acid method with glucose as standard (DuBois et al., 1956). A sulfuric acid-carbazole method was used to determine the total uronic acid content of CP80-1 using glucuronic acid as a standard. (Bitter & Muir, 1962). Total protein content of CP80-1 was determined by the Bradford method (Bradford, 1976).

2.3.2. Molecular weight determination

The molecular weight of CP80-1 was determined using Agilent 1200 high-performance gel permeation chromatography (HPGPC, Agilent, USA). The chromatographic column was TSK-GEL G4000PW (TOSOH Co., Tokyo, Japan), and the detector was Agilent differential refractive detector. Proteoglycan solution (20 μL, 5 mg/mL) was injected and eluted with sodium acetate (3 mmol/L) at 0.5 mL/min. Dextran standards (10, 40, 70, 500, 2000kDa) were used for calibration, and the relative molecular weight of CP80-1 was calculated from the standard curve.

2.3.3. Identification of monosaccharide composition

The monosaccharide composition of CP80-1 was determined by gas chromatography (GC) (Agilent, 4890D, Agilent Technologies, USA). 10 mg CP80-1 was hydrolyzed with sulfuric acid (2 mol/L, 5 mL) at 100°C for 8 h. After neutralization and concentration, it was reacted with hydroxylamine hydrochloride (10 mg) in pyridine solution (1 mL) at 90°C for 30 min. And then 1 mL acetic anhydride was added to the system and further reacted at 90°C for another 30 min. After the treatment, CP80-1 was converted into the corresponding derivatives of glyconitrile acetate for GC analysis by an HP-1 capillary column (0.25 mm × 60 m × 0.25 μm), 130°C held for 3 min, and then increased from 130 to 250°C at 4°C/min.

2.3.4. Ultraviolet (UV) spectroscopy analysis

The ultraviolet spectra of CP80-1 (0.1 mg/mL) were recorded in the wavelength range of 190–400 nm using a UV-2102 PCS spectrophotometer (Shimadzu Co., Japan).

2.3.5. Fourier-transform infrared spectroscopy (FT-IR) analysis

The infrared profile of CP80-1 was analyzed by FT-IR at 4000–500 cm⁻¹ (Nicolet Nexus 670, Thermo Electron Co., MA, USA).

2.4. Immunomodulatory activity of CP80-1

2.4.1. Cell culture

RAW264.7 cells were incubated in an incubator (Thermo, USA) at 37°C with 5% CO₂ and cultured in DMEM containing 10% heat-inactivated FBS. When the cell density was about 80%, subculture was performed.

2.4.2. Cells treatment

The endotoxin level of CP80-1 was detected by polymyxin B (PMB) (Xiao-Xiao et al., 2017). After preincubation, RAW264.7 cells (5 × 10⁵ cell/mL) were treated with PMB (100 μg/mL), LPS (1 μg/mL) and DMEM for 30 min. The LPS treatment group was used as a positive control, DMEM treatment group was blank control (CON) group.

2.4.3. Cell viability assay

After pre-incubation, the RAW264.7 cells (5 × 10⁴ cells/mL) were treated with different concentrations of CP80-1 (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μg/mL) or LPS (1 μg/mL). After being treated with CP80-1 and LPS for 24 h, 5 mg/mL of MTT solution was mixed into the culture medium, and the incubation was continued for another 4 h. At last, the absorbance of the solution was detected at 490 nm by the microplate reader (Molecular Device, USA).

2.4.4. Phagocytosis assay

The phagocytic capability of macrophages was detected by neutral assay. After pre-incubation, the cells (5 × 10⁴ cells/mL) were treated with CP80-1 solution (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μg/mL) or 1 μg/mL LPS for 24 h. After that, transfer 100 μL of 0.1% neutral red solution was to each well and incubate for an additional 30 min Cells were washed three times after culture, and cell lysis buffer was added into each well, and fully lysed at 4°C. The absorbance of the solution was determined at 540 nm (Molecular Device, USA).

2.4.5. Cell morphology observation

RAW264.7 cells were incubated in 6 well plates to 5 × 10⁵ cells per well, and then been treated with 2 mL of medium, LPS (1 μg/mL) or CP80-1 solution (0, 25, 50, 100, 200 μg/mL), respectively. The cells were cultured at 37°C for 24 h. After culture, cell morphology was observed with an inverted microscope (Nikon, Japan) and photographed.

2.4.6. Determination of NO, TNF-α, IL-1β and IL-6 secretion

After 24 h of stimulation by CP80-1 solution (0, 25, 50, 100, 200 μg/mL) or LPS (1 μg/mL), RAW264.7 cells culture supernatants of the collected and determined according to the instructions of NO assay kit and mouse TNF-α, IL-1β, IL-6 ELISA kits (Shanghai Yu Bo Biotech Co., Ltd, China).

2.5. Inhibition of Dectin-1 receptor using specific inhibitor

2.5.1. Determination of NO, TNF-α and IL-6 secretion

Laminarin was used as Dectin-1 receptor inhibitor. Cells were incubated in 12 well plates with 5 × 10⁵ cells/mL. CP80-1 group was added with 1 mL CP80-1 solution (100 μg/mL); CON and LPS groups were added with 1 mL medium and 1 mL LPS solution (1 μg/mL), respectively. Inhibitor pretreatment group was first treated with 1 mL laminarin solution (100 μg/mL), and then treated with 1 mL CP80-1 solution (100 μg/mL) or medium or LPS solution (1 μg/mL), respectively; Cells in each group were cultured at 37°C for 24 h. After that, supernatant was collected to determine NO, TNF-α and IL-6 using commercial kits as above.

2.5.2. Determination of mRNA expression levels of iNOS, TNF-α and IL-6 genes

The total RNA of RAW264.7 cells was extracted by Trizol. The cDNA was obtained using total RNA as a template in accordance with the kit's instructions. Using a Light Cycler 96 Real-Time PCR equipment and the RT–PCR kit from Roche (Switzerland), the expression levels of the genes iNOS, TNF-α, and IL-6 were determined (Roche, Switzerland). Their relative expression was calculated using the 2^-△△CT approach.

2.5.3. Western blot analysis

RAW264.7 cells were incubated in 6 well plates with 2 × 10⁶ cells per well, and the experimental method and groups were the same as the 2.5.1 section. After treatment, the total protein of RAW264.7 cells was extracted by RIPA buffer including protease and phosphatase inhibitors. The protein expression levels of Dectin-1, Syk, IKKα/β, p-IKKα/β, IκBα and β-actin were analyzed by western blot analysis. ECL chemiluminescence reagent was used to observe the protein bands, and the Clinx chemiluminescence imaging system was used for analysis (Shanghai, China). The image was examined using the Image J program.

2.5.4. Immunofluorescence assay

Nuclear translocation of the p65 subunit in RAW264.7 cells caused by CP80-1 was detected via immunofluorescence assay. RAW264.7 cells (5 × 10⁵ cells/mL) were treated with LPS (1 μg/mL), CP80-1 (100 μg/mL) or DMEM for 24 h, respectively. NF-κB activation-nuclear transport assay kit was used to detect the nuclear translocation of the p65 subunit. Images were analyzed by a fluorescence microscope (Nikon, Japan).

2.5.5. Statistical analysis

All data were expressed as mean ± SD, and their inter-group difference with one-way analysis of variance was conducted by SPSS 20.0 software. A value of P < 0.05 was taken as statistically significant.

3. Results and discussion

3.1. Characterization of CP80-1

3.1.1. Isolation and purification

In this study, the crude polysaccharides of C. cicadae were obtained by DEAE-cellulose column (Figure 1A), and then purified via Sephadex G-100 column (Figure 1B). The yield of CP80-1 from crude polysaccharides was 25.27%, total carbohydrate was 78.80%, total uronic acid was 3.81%, and protein was not detected.

Figure 1. Purified polysaccharide CP80-1 on DEAE-cellulose column (A) and Sephadex G-100 column (B). (C) The molecular weight of CP80-1 was determined by HPGPC. (D) The monosaccharide composition of CP80-1 was analyzed by GC (1-rhamnose, 2-arabinose, 3-xylose, 4-mannose, 5-glucose, and 6-galactose). (E) UV spectrum of CP80-1. (F) IR spectrum of CP80-1.

3.1.2. Molecular weight and monosaccharides composition determination

CP80-1 was shown a single and symmetrical peak on the HPGPC spectrum (Figure 1C), indicating that CP80-1 is homogeneous to some extent. According to the standard curve (Y = 38.32–4.116X, X–lgMw, Y–t; R = 0.995), the molecular weight of CP80-1 was calculated as 250.43 kDa. The HPLC chromatograms of standard monosaccharides and hydrolysates of CP80-1 were shown in Figure 1D. CP80-1 mainly consisted of glucose, xylose, and a small amount of rhamnose, and the molar ratio of CP80-1 was 19.04: 8.73: 1.00 according to the peak area shown in the HPGPC spectrum. It is well-known that the structure of Cordyceps polysaccharides is complex, and even polysaccharides extracted from the same raw material may have different structures. Two heteropolysaccharides with molecular weights of 30.9 kDa and 555.3 kDa were isolated from Isaria cicadae Miquel (i.e. C. cicadae), which consist of glucose, mannose and galactose in the ratios of 1.70:1.37:1.00 and 5.41:1.04:1.00, respectively (Xu et al., 2018). Besides, a polysaccharide isolated from C. militaris also consisted mainly of rhamnose, xylose and glucose, with a molecular weight of 28 kDa (Zhu et al., 2016).

3.1.3. UV and FT-IR spectrometric analysis

UV spectrum (Figure 1E) showed that CP80-1 had no UV absorption at 260 and 280 nm, indicating that contents of nucleic acids and proteins in CP80-1 were extremely low. Also, there was obvious terminal absorption at about 200 nm, showing the characteristic absorption peak of carbohydrates (Pan et al., 2012). FT-IR spectrum (Figure 1F) showed that there was a wide and strong absorption band at 3411 cm⁻¹, which was O–H stretching vibration. The weak peaks at 2927 and 1461 cm⁻¹ were respectively assigned to the stretching and angular vibration of C–H, both of which were characteristic absorption peaks of carbohydrates (Wang et al., 2017a; Zhang et al., 2018). The bands between 1600–1650 cm⁻¹ and 1400–1300 cm⁻¹ corresponded to C–O stretching vibration (Pan et al., 2012). In addition, the absorption bands at 1060, 848 and 892 cm⁻¹ indicated that both α and β-configuration glycosidic bonds existed in CP80-1, while there had no characteristic absorption peaks of mannitol (Barker et al., 1954).

3.2. Immunomodulatory activities of CP80-1

3.2.1. Effect of CP80-1 on cell viability

Before studying the immunomodulatory activity of CP80-1, it is necessary to determine its endotoxin contamination and cytotoxicity. The results showed that PMB treatment reduced LPS-induced NO secretion in RAW264.7 cells, but did not affect the CP80-1 treatment group (100 μg/mL), indicating that CP80-1 almost had no endotoxin contamination (Figure 2A). As can be shown in Figure 2B, the cell viability of LPS group was considerably higher (P 0.05) than that of the CON group. After the treatment of CP80-1 (3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μg/mL), the cell viability was increased at first, then decreased, reaching a maximum at a concentration of 25 μg/mL. Therefore, between 3.125–400 μg/mL, CP80-1 showed no cytotoxicity and significantly enhanced cell viability, and experimental concentration could be selected from this range for subsequent experiments.

Figure 2. Endotoxin contamination test using PMB in CP80-1-treated RAW264.7 cells (A) and effect of CP80-1 on cell viability (B) and phagocytosis (C). All values are expressed as the mean ± SD (n = 5). Different letters indicate significant differences (P < 0.05).

3.2.2. Effect of CP80-1 on phagocytosis of RAW264.7 cells

Macrophages possess an active phagocytic function, which could phagocytize foreign antigens, playing a significant role in the immunoreaction of the body (Liu et al., 2017; Zhong et al., 2016). According to Figure 2C, CP80-1 boosted RAW264.7 cells’ phagocytosis in a concentration-dependent manner (3.125–200 μg/mL). Of note, the cell phagocytosis ratio reached 171.25% at 200 μg/mL, which was close to 188.94% of the LPS group, suggesting that CP80-1 could promote the phagocytic activity of macrophages.

3.2.3. Effect of CP80-1 on cell morphology

Figure 3 showed that RAW264.7 cells in the CON group were round, small and in a resting state. However, after being treated with LPS (positive control, 1 μg/mL) or CP80-1 (25–200 μg/mL), the cell morphology was changed to a shuttle shape, increased in size, and some cells grew pseudopods. Moreover, these changes became progressively more apparent with the increasing concentrations of CP80-1. These results suggest that RAW264.7 cells can be activated and change cell morphology after being treated with CP80-1.

Figure 3. Effect of CP80-1 on RAW264.7 cell morphology. RAW264.7 cells were cultured with CP80-1 solution in different concentrations 0 μg/mL (A), 25 μg/mL (B), 50 μg/mL (C), 100 μg/mL (D), 200 μg/mL (E) or LPS (positive control, 1 μg/mL) (F) for 24 h.

3.2.4. Effect of CP80-1 on NO, TNF-α, IL-1β and IL-6 cytokine secretion

When RAW264.7 cells are stimulated, small molecules of NO and inflammatory cytokines (TNF-α, IL-1β and IL-6, etc) for inflammatory and immune response are secreted. These cytokines can directly fight against microorganisms and tumor cells, and also trigger the immune chain reaction of the body, which play a key role in the activation and regulation of macrophages (Liu et al., 2017). As shown in Figure 4, the secretion of cellular NO, TNF-α, IL-1β, and IL-6 was significantly increased when treated with CP80-1 compared to the CON group (25, 50, 100, and 200 μg/mL) (P < 0.05). When the concentration of CP80-1 was 100 and 200 μg/mL, the secretion of NO in the treatment groups shows no significant differences with the LPS group (P > 0.05). These results showed that CP80-1 from C. cicadae polysaccharides can activate RAW264.7 cells and promote the secretion of cytokines, which were consistent with the immunomodulatory activity of other polysaccharides from fungal food or medicines, such as Ganoderma lucidum (Guo et al., 2009), C. militaris (Lee et al., 2015), Smilax glabra Roxb. (Wang et al., 2017b).

Figure 4. Effect of CP80-1 on NO (A), TNF-α (B), IL-1β (C), and IL-6 (D) secretion in RAW264.7 cells. All values are expressed as the mean ± SD (n = 5). Different lowercase alphabet letters (a–d) indicate significant differences (P < 0.05).

3.3. The possible mechanism of the immunomodulatory activity of CP80-1 in RAW 264.7 cells

3.3.1. Effect of CP80-1 on the secretion and mRNA expression of cytokines in RAW264.7 cells via inhibiting Dectin-1 receptor

There are many kinds of specific polysaccharide receptors on the surface of macrophages such as the Dectin-1. By binding with these receptors, polysaccharides could activate the downstream signal pathways and macrophages, which then induce immune responses (Dennehy & Brown, 2007). Many cellular responses, including phagocytosis, autophagy and the secretion of many cytokines and chemokines, are regulated by Dectin-1 signaling (Dambuza & Brown, 2015). In the present study, we preliminarily explored the function of the Dectin-1 receptor in the activation of CP80-1-treated RAW264.7 cells. It showed that after pretreatment of RAW264.7 cells with Dectin-1 receptor inhibitor (100 μg/mL), the secretion levels of NO, TNF-α and IL-6 were all decreased in RAW264.7 cells compared with the CON group. Among them, the secretion levels of NO and TNF-α decreased more significantly (P < 0.05) (Figure 5). In addition, the mRNA expression levels of iNOS, TNF-α, and IL-6 genes were down-regulated in RAW264.7 cells when the Dectin-1 receptor was inhibited, which were largely consistent with the trend of decreased in cytokine secretion. These data further showed that CP80-1 may regulate the secretion of cytokines by regulating the expression of genes (iNOS, TNF-α, and IL-6) and that these effects were related to the Dectin-1 receptor. NO is recognized as an intercellular messenger and participates in signal transduction and immune regulation, and could modulate the production of cytokines, such as IL-6, TNF-α, and IL-1β (Wu et al., 2013). At the same time, cytokines also play a feedback regulation on NO in macrophages. Therefore, the changing trend of NO secretion of RAW 264.7 cells was not completely consistent with that of IL-6, TNF-α, and IL-1β after CP80-1 treatment.

Figure. 5. Effect of Dectin-1 receptor inhibitor laminarin on CP80-1-induced cytokines secretion and mRNA expression in RAW264.7 cells. NO (A), TNF-α (B), and IL-6 secretion (C) in RAW264.7 cells; iNOS (D), TNF-α (E), and IL-6 (F) mRNA expression in RAW264.7 cells. All values are expressed as the mean ± SD (n = 5). Different lowercase alphabet letters (a–f) indicate significant differences (P < 0.05).

3.3.2. Effect of CP80-1 on Dectin-1 receptor-mediated Syk/NF-κB signaling pathway in RAW 264.7 cells

In our study, the related protein expression of Syk/NF-κB signaling pathway in RAW264.7 cells was detected by western blot analysis after Dectin-1 receptor inhibited. As shown in Figure 6, the addition of laminin dramatically reduced the expression level of the Dectin-1 receptor in RAW264.7 cells in the CON group (P < 0.05). Additionally, the expression level was restored following treatment with CP80-1 and LPS, and it did not substantially differ from the group that did not receive any laminin intervention (P > 0.05). Laminarin bind to the Dectin-1 receptor without triggering the downstream signaling pathway of RAW 264.7 cells, so it has little effect on the expression of the Dectin-1 receptor in cells. However, when the Dectin-1 receptor was inhibited, polysaccharides and LPS could stimulate the up-regulation of Dectin-1 receptor expression, which is agree with the reported study (Wang et al., 2014b). The expression of Syk and p-IKKα/β protein in cells was considerably higher in CP80-1treatment group (100 g/mL) compared with the CON group (P < 0.05); while pretreatment with laminarin (100 μg/mL) for 30 min caused a decrease in CP80-1-induced protein expression of Syk and p-IKKα/β (P < 0.05), and of IκBα was showed to be the opposite trend.

Figure 6. Effect of CP80-1 on Dectin-1 receptor-mediated Syk/NF-κB signaling pathway in RAW264.7 cells. (A) Western blot of related proteins. (B) Gray scale analysis of protein bands by Image J software. All values are expressed as the mean ± SD (n = 5). Different lowercase alphabet letters (a–f) indicate significant differences (P < 0.05).

NF-κB proteins are usually homodimer/heterodimer formed by the binding of p65 and p50 subunits (Gao et al., 2016). In the resting state of the cell, the NF-κB protein binds to the inhibitory protein IκB in the cytoplasm to form a trimer (Sang et al., 2020). When the upstream signaling factor is delivered, the downstream IKK kinase is activated and then phosphorylates the IκB protein, causing it to depolymerize from the trimer. The p65 subunit of the dimer rapidly enters the nucleus and binds to the specific sequences of nuclear DNA to initiate transcription of the relevant genes, and the intracellular NF-κB pathway is then activated (Lawrence et al., 2005; Zozo et al., 2021). Our results showed that CP80-1 (100 μg/mL) could promote the expression of Syk and the phosphorylation of IKKα/β, as well as the degradation of IκBα proteins in RAW264.7 cells, but these effects were diminished when the Dectin-1 receptor was inhibited by laminarin (100 μg/mL). These results suggested that the activation of RAW 264.7 cells by CP80-1 was closely related to the Syk/NF-κB signaling pathway mediated by the Dectin-1 receptor.

3.3.3. Effect of CP80-1 on nuclear translocation of NF-κB

Figure 7 showed that the majority of p65 subunits in the CON group were present in the cytoplasm. After being treated with CP80-1, the fluorescence of p65 subunit was enhanced, and mostly overlapped with the fluorescence of nucleus. Based on the immunofluorescence pictures, CP80-1 and LPS groups fluoresced significantly more than the CON group, which indicated a translocation of most of the p65 subunits into the nucleus. (P < 0.01). These results indicated that most of the p65 subunits translocated into the nucleus NF-κB signaling pathway were activated in RAW264.7 cells, which was consistent with the above results.

Figure 7. Effect of CP80-1 on NF-κB activation in RAW264.7 cells. (A) Immunofluorescence assay. (B) Quantifications of the immunofluorescence pictures (P < 0.01). Images were captured by a fluorescence microscope (× 10 μm).

Several species, including C. militaris, Ophiocordyceps Sinensis and C. cicadae, all belong to the genus Cordyceps, have been reported to possess immunomodulatory properties, and the underlying mechanisms have been investigated. C. militaris polysaccharide is reported to activate macrophages through the MAPKs and NF-κB signaling pathways associated with multiple receptors, including the Dectin-1 receptor (Lee et al., 2015). Meanwhile, polysaccharides of the liquid culture of O. Sinensis activate RAW264.7 cells for an improvement of immunomodulation activities via MAPK and PI3 K/Akt signaling pathways (Liu et al., 2021). Besides, a polysaccharide of Isaria cicadae Miquel is reported to activate RAW264.7 cells through the TLR4/MAPK/NF-κB signaling pathway (Xu et al., 2020). Therefore, the current study looked at the mechanism of Cordyceps’ immunomodulatory function from the standpoint of the Dectin-1 receptor. Apparently, our study reconfirmed that Dectin-1 receptor is important for activation of the macrophage by polysaccharides of C. cicadae through Syk/NF-κB signaling pathway, and the schematic diagram was showed in Figure 8.

Figure 8. Dectin-1 receptor-mediated Syk/NF-kB signaling pathway with CP80-1 treatment.

Reports show that polysaccharides between 400 and 5 kDa exhibit the most potent macrophage-activating activity, as polysaccharides of this molecular weight not only has good permeability, but also forms active polymer structures (Im et al., 2005), and CP80-1 is exactly in this range. In addition, it is generally considered that polysaccharides with β configuration have the higher activity, and CP80-1 has both α and β-configuration glycosidic bonds (Wang et al., 2022). The immunomodulatory effects of polysaccharides are affected by their molecular weight, monosaccharide composition, α/β-configuration, conformation, glycosidic linkage, branching degree, and even their advanced structures (Yin et al., 2021). Therefore, the relationship between the structure of CP80-1 and its mechanism of immunomodulatory activity remains to be further explored.

4. Conclusion

In this study, a polysaccharide CP80-1 with a molecular weight of 250.431 kDa, composed of glucose, xylose and rhamnose, was isolated from C. cicadas. CP80-1 was found to exert immunomodulatory effects of RAW264.7 cells via promoting proliferation and phagocytosis, increasing the release of immune factors (NO, TNF-α, IL-1β, IL-6). In addition, our study further confirmed that CP80-1 activated macrophages in association with Dectin-1 receptor-mediated downstream Syk/NF-kB signaling pathway. This study provides evidence for CP80-1 as a natural immune enhancing agent and provides a reference for the development and utilization of medicinal materials resources of C. cicadae.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

Additional information

Funding

This work was supported by National Key Research and Development Program of China: [Grant Number 2018YFC1706105] and Key Project at Central Government Level: [Grant Number 2060302].