2022, Vol. 26 ›› Issue (31): 5040-5046

Effect of phosphocreatine modified chitosan hydrogel on polarization and inflammatory factor expression in rat bone marrow-derived macrophages

Sheng Weibei1, 2, 3, Xiong Ao2, 3, Liu Su2, 3, Deng Jiapeng2, 3, Weng Jian2, 3, Yu Fei2, 3, Chen Yingqi2, 3, Zeng Hui2, 3   

Abstract: BACKGROUND: Macrophage polarization-mediated inflammation plays an important role in chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. Phosphocreatine-modified chitosan hydrogels show good potential application prospects in tissue repair, but the effect of this new hydrogel on polarization and inflammatory factor expression in macrophages is still unclear.
OBJECTIVE: To explore the effect of phosphocreatine modified methacrylic anhydride chitosan hydrogel on macrophage polarization and inflammatory factor expression.
METHODS: One-step freeze-drying method was used to prepare phosphocreatine modified methacrylic anhydride chitosan with good water solubility. Hydrogels were further prepared under low toxicity initiators and ultraviolet irradiation. Bone marrow-derived macrophages were extracted from SD rats and cultured for 7 days, and then divided into three groups. Complete cell culture medium was added in the control group. Complete cell culture medium containing lipopolysaccharide was added in the lipopolysaccharide group. Freeze-dried hydrogel samples and cell complete medium containing lipopolysaccharide were added in the chitosan hydrogel group. Cell viability was detected using CCK-8 assay. RT-PCR, ELISA, western blot assay, and immunofluorescence staining were used to detect macrophage polarization and the expression of inflammatory factors.
RESULTS AND CONCLUSION: (1) CCK-8 assay showed that after 1, 3, and 5 days of culture, there was no significant difference in the cell viability among the three groups (P > 0.05). (2) Western blot assay and immunofluorescence staining showed that compared with the control group, the M2 macrophage surface marker CD206 protein expression was down-regulated (P < 0.05) and the ARG1 protein expression was up-regulated (P < 0.05) in the lipopolysaccharide group after 1 and 3 days of culture. Compared with the lipopolysaccharide group, the protein expression levels of CD206 and ARG1 in the chitosan hydrogel group were up-regulated after 3 days of culture (P < 0.05). (3) ELISA and RT-PCR results showed that compared with the control group, the protein and mRNA expression levels of pro-inflammatory factors interleukin-1β and interleukin-6 in the lipopolysaccharide group increased after 1 and 3 days of culture (P < 0.05). Compared with the lipopolysaccharide group, the protein and mRNA expression levels of interleukin-1β and interleukin-6 in the chitosan hydrogel group were decreased after 1 and 3 days of culture (P < 0.05). (4) These results have shown that phosphocreatine modified methacrylic anhydride chitosan hydrogel promotes M2-type polarization of macrophages and inhibits the expression of pro-inflammatory factors, which may provide a potential treatment for immune-related diseases in the future.
Key words: chitosan hydrogel, bone marrow-derived macrophage, polarization, inflammatory factor, lipopolysaccharide