Effects of Siglec-E on parthenolide inhibiting microglia M1 polarization and targeting MAPK/NF-κB pathway

Abstract

Objective To explore the impact of the sialic acid binding lectin-E (Siglec-E) on the inhibitory properties of parthenolide(PTL) against lipopolysaccharide (LPS)-induced M1 polarization of microglia(BV2). Methods ① Single cell sequencing data of Siglece related mouse brain tissue was obtained from Gene Expression Omnibus (GEO) database and divided into the WT group (n=3) and the Siglece-/- group (n=4). The microglia cells were screened, and the enrichment analysis was performed to analyze related differential genes and pathways. BV2 cells were constructed by the shRNA interference technique and were divided into NC-shRNA and Siglece-shRNA to detect the expression level of Siglec-E (Siglece). ② NC-shRNA and Siglece-shRNA cells were respectively divided into the Control group, LPS group, PTL group and PTL+LPS group (n=3). The mRNA levels of markers of M1 polarization in microglia, iNOS, IL-1β and IL-6, were detected by RT-qPCR. Siglecefl/fl and Cx3cr1cre mice were mated to obtain microglia-specific Siglece deletion (Siglecefl/fl×Cx3cr1cre) mice, and LPS-induced neuroinflammation model was established. ③ Nine WT and Siglecefl/fl×Cx3cr1cre male mice were assigned to the Control group, LPS group and PTL+LPS group (n=3). RT-qPCR, immunofluorescence assay and Western blotting were used to verify the knock-out effect and polarization-related pathways, and to investigate the mechanism of Siglec-E affecting PTL inhibition of M1 polarization of microglia. Results Compared with the NC-shRNA group, the expression of Siglec-E in the Siglece-shRNA group was significantly decreased (P < 0.01), indicating that the Siglec-E knock-down cell model was successfully established. With the stimulation of LPS, mRNA levels of iNOS, IL-1β and IL-6 were significantly up-regulated compared with the Control group both in shRNA cells and Siglece -shRNA cells (P < 0.01). With the influence of PTL and LPS, the markers of M1 polarization in NC-shRNA cells mentioned before were significantly decreased (P < 0.05), while for Siglice-shRNA cells, there were no significant changes in the markers of M1 polarization. PTL inhibited the phosphorylation of JNK and IκB protein (P < 0.01) and the nuclear translocation of NF-κB in BV2 cells, down-regulated Siglec-E, and weakened the inhibitory effect. Compared with mice in the WT group, the expression of Siglec-E in microglia of Siglecefl/fl×Cx3cr1cre mice was decreased significantly (P < 0.01), and the inhibitory effect of PTL on the phosphorylation of NF-κB in microglia of Siglecefl/fl×Cx3cr1cre mice was also decreased. Conclusion The absence of Siglec-E in microglia attenuates the inhibition of M1 polarization by the MAPK/NF-κB pathway targeted by PTL.

Keywords

• sialic acid binding lectin e
• microglia
• parthenolide
• m1 polarization