Structural Characteristics of Sarcomyxa edulis Polysaccharide and Its Mechanism of Regulating Macrophage Immunomodulatory Activity

doi:10.13560/j.cnki.biotech.bull.1985.2023-0483

Abstract

Abstract:

【Objective】In order to clarify the structural characteristics of Sarcomyxa edulis polysaccharides and explore its mechanism of regulating macrophage immunomodulatory activity. 【Method】The crude polysaccharide of S. edulis was obtained by water extraction and ethanol precipitation, and the purified polysaccharide component SEP-0a was obtained by DEAE-52 ion exchange column chromatography and Sephacryl S-400 glucan gel column chromatography. The physicochemical properties of SEP-0a were investigated by high performance gel permeation chromatography evaporative light-scattering detector, ion exchange chromatography high performance liquid chromatography and Fourier transform infrared spectrometer. On this basis, the immunomodulatory effect of polysaccharide SEP-0a from S. edulis on macrophage cell RAW264.7 was further studied by CCK-8, ELISA, qPCR, Western Blot and other methods. 【Result】The S. edulis polysaccharide SEP-0a was composed of galactose, mannose, glucose and fucose with a relative molecular weight of 4.23×10⁴ Da, and had α and β configurations. The results of in vitro immunocompetence showed that S. edulis polysaccharide SEP-0a significantly increased the proliferation and phagocytic ability of RAW264.7 cells, promoted the secretion of reactive oxygen species（ROS）, nitric oxide（NO）and increased the expressions of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, and IL-6 in these cells. Western blot results showed that this polysaccharide significantly increased p65 protein phosphorylation expression in the nuclear factor-κB（NF-κB）signaling pathway. 【Conclusion】The polysaccharide SEP-0a of S. edulis may regulates the immune activity of macrophages by NF-κB signal pathway.

Key words: Sarcomyxa edulis polysaccharide, structural characteristics, RAW264.7 cells, NF-κB signal pathway

WANG Zi-xuan, SUI Yu, SHANG Xue-yu, MA Si-jia, WU Tian-xiang, LIU Yang, WANG Qi. Structural Characteristics of Sarcomyxa edulis Polysaccharide and Its Mechanism of Regulating Macrophage Immunomodulatory Activity[J]. Biotechnology Bulletin, 2024, 40(1): 308-321.

Figures/Tables 15

Table 1 Primer sequences

基因 Gene	上游Forward primer（5'-3'）	下游Reverse primer（5'-3'）
TNF-α	CCCTCACACTCAGATCATCTTCT	GCTACGACGTGGGCTACAG
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
IL-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

Table 2 Primer sequences

基因 Gene	上游 Forward primer（5'-3'）	下游 Reverse primer（5'-3'）
NO	CTGGTGAAGGAACGGGTCAG	CCGATCATTGACGGCGAGAAT
TNF-α	CCCTCACACTCAGATCATCTTCT	GCTACGACGTGGGCTACAG
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
IL-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

Fig. 1 Elution curve of S. edulis polysaccharide A: DEAE-52 ion exchange column chromatography; B: sephacryl S-400 glucan gel column chromatography

Fig. 2 Elution curve of SEP-0a high performance gel permeation chromatography

Fig. 3 SEP-0a ion chromatogram A: Standard sample; B: SEP-0a

Fig. 4 Infrared spectrum of SEP-0a

Fig. 5 Effects of SEP-0a at different concentrations on the proliferation of RAW264.7 cells **P<0.01, compared with blank group（n=6）

Fig. 6 Effects of SEP-0a at different concentrations on the phagocytosis of RAW264.7 cells *P<0.05; **P<0.01, compared with blank group（n=3）

Fig. 7 Effects of SEP-0a at different concentrations on the ROS secretion of RAW264.7 cells **P<0.01, compared with blank group（n=6）

Fig. 8 Effects of SEP-0a at different concentrations on NO secretion by RAW264.7 cells **P<0.01, compared with blank group（n=4）

Fig. 9 Effects of SEP-0a at different concentrations on TNF-α（A）, IL-1β（B）and IL-6（C）secretion by RAW264.7 cells **P<0.01, compared with blank group（n=4）

Fig. 10 Effects of SEP-0a at different concentrations on TNF-α（A）, IL-1 β（B）and IL-6（C）gene expressions by RAW264.7 cells **P<0.01, compared with blank group（n=4）

Fig. 11 Effects of SEP-0a on protein phosphorylation in RAW264.7 cells **P<0.01, compared with blank group

Fig. 12 Effects of NF-κB inhibitors on the secretions of NO（A）, TNF-α（B）, IL-1β（C）and IL-6（D）induced by SEP-0a in RAW264.7 cells **P<0.01, compared with blank group; ##P<0.01, compared with SEP-0a（n=3）

Fig. 13 Effects of NF-κB inhibitors on SEP-0a on the gene expressions of NO（A）, TNF-α（B）, IL-1β（C）and IL-6（D）by RAW264.7 cells **P<0.01, compared with blank group; ##P<0.01, compared with SEP-0a（n=4）

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