Cirsii Herba glycoprotein promotes macrophage M1 polarization through MAPK and NF-κB signaling pathways via interaction with TLR4.

The present study aimed to extract and purify the glycoprotein from Cirsii Herba (CHPs), and investigate its immunomodulatory activity and molecular mechanism in RAW264.7 macrophages. The results showed that CHPs contained 14.8 % carbohydrates and 80.4 % proteins. CHPs were identified as a glycoprotein around 70 kDa and contained 17 different amino acids, in which the Glu and Asp were predominant. The carbohydrate chain in CHPs was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose with the molecular ratio of 6.387: 24.358: 5.766: 8.877: 12.098: 20.427: 7.090: 14.997. CHPs significantly boosted pinocytic and phagocytic activities, increased the secretions of inflammatory factors (NO, TNF-α and IL-6) and chemokines (CXCL2 and CXCL10), and promoted the expressions of accessory and costimulatory molecules (CD40, CD80, CD86, MHC I and MHC II). RNA-seq analysis identified 721 DEGs, 1575 GO terms and 69 KEGG pathways. The pathway inhibition assay presented that MAPK and NF-κB pathways were essential to macrophage activation by CHPs. TLR4 was revealed as a functional receptor and involved in the early recognition of CHPs. These results indicated that CHPs as a glycoprotein promoted macrophage polarization to M1 phenotype mainly via TLR4-dependent MAPK and NF-κB pathways.